Transgenic Plants With Enhanced Agronomic Traits

ABSTRACT

This invention provides recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. Also provided by this invention is transgenic seed for growing a transgenic plant having recombinant DNA in its genome and exhibiting an enhance agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold stress and/or improved seed compositions. Also disclosed are methods for identifying such transgenic plants by screening for nitrogen use efficiency, yield, water use efficiency, growth under cold stress, and seed composition changes. This invention also discloses a method of identifying the target genes of a transcription factor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S.provisional application Ser. No. 60/713,150, filed Aug. 30, 2005, andInternational application PCT/US2006/033989, both incorporated herein byreference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computerreadable form (CRF) of the sequence listing, all on CD-Rs, eachcontaining the text file named“38-21(53948)A-_seqListing_amended_(—)02182008.txt”, which is 31.6 MB(measured in MS-WINDOWS) and was created on Feb. 28, 2008 areincorporated herein by reference.

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics anddevelopmental biology. More specifically, the present inventions providetransgenic seeds for crops, wherein the genome of said seed comprisesrecombinant DNA, the expression of which results in the production oftransgenic plants with enhanced agronomic traits.

SUMMARY OF THE INVENTION

This invention employs recombinant DNA for expression of proteins thatare useful for imparting enhanced agronomic traits to the transgenicplants. Recombinant DNA in this invention is provided in a constructcomprising a promoter that is functional in plant cells and that isoperably linked to DNA that encodes a protein having at least one aminoacid domain in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam domain names as identified in Table 11.In more specific embodiments of the invention the protein expressed inplant cells has an amino acid sequence with at least 90% identity to aconsensus amino acid sequence in the group of consensus amino acidsequences consisting of the consensus amino acid sequence constructedfor SEQ ID NO: 194 and homologs thereof listed in Table 7 through theconsensus amino acid sequence constructed for SEQ ID NO: 386 andhomologs thereof listed in Table 7. In even more specific embodiments ofthe invention the protein expressed in plant cells is a protein selectedfrom the group of proteins identified in Table 1.

Other aspects of the invention are specifically directed to transgenicplant cells comprising the recombinant DNA of the invention, transgenicplants comprising a plurality of such plant cells, progeny transgenicseed, embryo and transgenic pollen from such plants. Such plant cellsare selected from a population of transgenic plants regenerated fromplant cells transformed with recombinant DNA and that express theprotein by screening transgenic plants in the population for an enhancedtrait as compared to control plants that do not have said recombinantDNA, where the enhanced trait is selected from group of enhanced traitsconsisting of enhanced water use efficiency, enhanced cold tolerance,increased yield, enhanced nitrogen use efficiency, enhanced seed proteinand enhanced seed oil.

In yet another aspect of the invention the plant cells, plants, seeds,embryo and pollen further comprise DNA expressing a protein thatprovides tolerance from exposure to an herbicide applied at levels thatare lethal to a wild type of said plant cell. Such tolerance isespecially useful not only as an advantageous trait in such plants butis also useful in a selection step in the methods of the invention. Inaspects of the invention the agent of such herbicide is a glyphosate,dicamba, or glufosinate compound.

Yet other aspects of the invention provide transgenic plants which arehomozygous for the recombinant DNA and transgenic seed of the inventionfrom corn, soybean, cotton, canola, alfalfa, wheat or rice plants.

In other important embodiments for practice of various aspects of theinvention, the plants of this invention can be further enhanced withstacked traits, e.g., a crop having an enhanced agronomic traitresulting from expression of DNA disclosed herein, in combination withherbicide, disease, and/or pest resistance traits.

This invention also provides methods for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith an enhanced trait resulting from expression of stably-integrated,recombinant DNA for expressing a protein having at least one domain ofamino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 11. Morespecifically the method comprises (a) screening a population of plantsfor an enhanced trait and a recombinant DNA, where individual plants inthe population can exhibit the trait at a level less than, essentiallythe same as or greater than the level that the trait is exhibited incontrol plants which do not express the recombinant DNA, (b) selectingfrom the population one or more plants that exhibit the trait at a levelgreater than the level that said trait is exhibited in control plants,(c) verifying that the recombinant DNA is stably integrated in saidselected plants, (d) analyzing tissue of a selected plant to determinethe production of a protein having the function of a protein encoded bynucleotides in a sequence of one of SEQ ID NO:1-193; and (e) collectingseed from a selected plant. In one aspect of the invention the plants inthe population further comprise DNA expressing a protein that providestolerance to exposure to an herbicide applied at levels that are lethalto wild type plant cells and the selecting is effected by treating thepopulation with the herbicide, e.g. a glyphosate, dicamba, orglufosinate compound. In another aspect of the invention the plants areselected by identifying plants with the enhanced trait. The methods areespecially useful for manufacturing corn, soybean, cotton, alfalfa,wheat or rice seed.

Another aspect of the invention provides a method of producing hybridcorn seed comprising acquiring hybrid corn seed from a herbicidetolerant corn plant which also has stably-integrated, recombinant DNAcomprising a promoter that is (a) functional in plant cells and (b) isoperably linked to DNA that encodes a protein having at least one domainof amino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 11. Themethods further comprise producing corn plants from said hybrid cornseed, wherein a fraction of the plants produced from said hybrid cornseed is homozygous for said recombinant DNA, a fraction of the plantsproduced from said hybrid corn seed is hemizygous for said recombinantDNA, and a fraction of the plants produced from said hybrid corn seedhas none of said recombinant DNA; selecting corn plants which arehomozygous and hemizygous for said recombinant DNA by treating with anherbicide; collecting seed from herbicide-treated-surviving corn plantsand planting said seed to produce further progeny corn plants; repeatingthe selecting and collecting steps at least once to produce an inbredcorn line; and crossing the inbred corn line with a second corn line toproduce hybrid seed.

Another aspect of the invention provides a method of selecting a plantcomprising plant cells of the invention by using an immunoreactiveantibody to detect the presence of protein expressed by recombinant DNAin seed or plant tissue. Yet another aspect of the invention providesanti-counterfeit milled seed having, as an indication of origin, a plantcell of this invention.

Still other aspects of this invention relate to transgenic plants withenhanced water use efficiency or enhanced nitrogen use efficiency. Forinstance, this invention provides methods of growing a corn, cotton orsoybean crop without irrigation water comprising planting seed havingplant cells of the invention which are selected for enhanced water useefficiency. Alternatively methods comprise applying reduced irrigationwater, e.g. providing up to 300 millimeters of ground water during theproduction of a corn crop. This invention also provides methods ofgrowing a corn, cotton or soybean crop without added nitrogen fertilizercomprising planting seed having plant cells of the invention which areselected for enhanced nitrogen use efficiency.

BRIEF DESCRIPTION OF FIGURES

FIG. 1-4 illustrate plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO:1-193 are nucleotide sequences of the coding strand of DNA for“genes” used in the recombinant DNA imparting an enhanced trait in plantcells, i.e. each represents a coding sequence for a protein;

SEQ ID NO:194-386 are amino acid sequences of the cognate protein of the“genes” with nucleotide coding sequence 1-193;

SEQ ID NO: 387-12580 are amino acid sequences of homologous proteins;

SEQ ID NO: 12581-12601 are nucleotide sequences of the elements in baseplasmid vectors

SEQ ID NO: 12602 is a consensus amino acid sequence.

SEQ ID NO: 12603 is a nucleotide sequence of a base plasmid vectoruseful for corn transformation; and

SEQ ID NO: 12604 is a nucleotide sequence of a base plasmid vectoruseful for soybean transformation.

SEQ ID NO: 12605 is a nucleotide sequence of a base plasmid vectoruseful for cotton transformation.

SEQ ID NO: 12606 is the nucleotide sequence of plasmid PMON17730.

SEQ ID NO: 12607 is the nucleotide sequence of PHE0010424_PMON17730.

SEQ ID NO: 12608 is the consensus sequence of SEQ ID NO: 371 and 11homologs

As used herein, a “transgenic plant” means a plant whose genome has beenaltered by the incorporation of exogenous DNA, e.g., by transformationas described herein. The term “transgenic plant” is used to refer to theplant produced from an original transformation event, or progeny fromlater generations or crosses of a plant so transformed, so long as theprogeny contains the exogenous genetic material in its genome.“Exogenous DNA” means DNA, e.g., recombinant DNA, originating from orconstructed outside of the plant including natural or artificial DNAderived from the host “transformed” organism of a different organism.

As used herein, “recombinant DNA” means DNA which has been a geneticallyengineered or constructed outside of a cell, including DNA containingnaturally occurring DNA or cDNA, or synthetic DNA.

As used herein, a “functional portion” of DNA is that part whichcomprises an encoding region for a protein segment that is sufficient toprovide the desired enhanced agronomic trait in plants transformed withthe DNA activity. Where expression of protein is desired, a functionalportion will generally comprise the entire coding region for theprotein, although certain deletions, truncations, rearrangements and thelike of the protein may also maintain, or in some cases improve, thedesired activity. One skilled in the art is aware of methods to screenfor such desired modifications and such functional portion of theprotein is considered within the scope of the present invention.

As used herein, “consensus sequence” means an artificial, amino acidsequence of conserved parts of the proteins encoded by homologous genes,e.g., as determined by a CLUSTALW alignment of amino acid sequence ofhomolog proteins.

As used herein, “homolog” means a protein in a group of proteins thatperform the same biological function, e.g., provide an enhancedagronomic trait in transgenic plants of this invention. Homologs areexpressed by homologous genes which are genes that encode proteins withthe same or similar biological function. Homologous genes may begenerated by the event of speciation (see ortholog) or by the event ofgenetic duplication (see paralog). Orthologs refer to a set ofhomologous genes in different species that evolved from a commonancestral gene by specification. Normally, orthologs retain the samefunction in the course of evolution; and paralogs refer to a set ofhomologous genes in the same species that have diverged from each otheras a consequence of genetic duplication. Thus, homologous genes can befrom the same or a different organism. Homologous DNA includes naturallyoccurring and synthetic variants. For instance, degeneracy of thegenetic code provides the possibility to substitute at least one base ofthe protein encoding sequence of a gene with a different base withoutcausing the amino acid sequence of the polypeptide produced from thegene to be changed. Hence, a polynucleotide useful in the presentinvention may have any base sequence that has been changed from SEQ IDNO:1 through SEQ ID NO: 193 by substitution in accordance withdegeneracy of the genetic code. Homologs are proteins which, whenoptimally aligned, has at least 60% identity (say at least 70% or 80% or90% identity) over the full length of a protein identified herein, or ahigher percent identity especially over a shorter functional part of theprotein, e.g., 70% to 80 or 90% amino acid identity over a window ofcomparison comprising a functional part of the protein imparting theenhanced agronomic trait. Homologs include proteins with an amino acidsequence that has at least 90% identity to a consensus amino acidsequence of proteins and homologs disclosed herein.

Homologs can be identified by comparison of amino acid sequence, e.g.,manually or by using known homology-based search algorithms such asthose commonly known and referred to as BLAST, FASTA, andSmith-Waterman. A local sequence alignment program, e.g., BLAST, can beused to search a database of sequences to find similar sequences, andthe summary Expectation value (E-value) used to measure the sequencebase similarity. As a protein hit with the best E-value for a particularorganism may not necessarily be an ortholog or the only ortholog, areciprocal query is used in the present invention to filter hitsequences with significant E-values for ortholog identification. Thereciprocal query entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal query's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. A further aspect of theinvention comprises functional homolog proteins which differ in one ormore amino acids from those of disclosed protein as the result ofconservative amino acid substitutions, e.g., substitutions are among:acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; basic (positively charged) amino acids such as arginine,histidine, and lysine; neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;amino acids having aliphatic side chains such as glycine, alanine,valine, leucine, and isoleucine; amino acids having aliphatic-hydroxylside chains such as serine and threonine; amino acids havingamide-containing side chains such as asparagine and glutamine; aminoacids having aromatic side chains such as phenylalanine, tyrosine, andtryptophan; amino acids having basic side chains such as lysine,arginine, and histidine; amino acids having sulfur-containing sidechains such as cysteine and methionine; naturally conservative aminoacids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the homologs encoded by DNAuseful in the transgenic plants of the invention are those proteinswhich differ from a disclosed protein as the result of deletion orinsertion of one or more amino acids in a native sequence.

As used herein, “transcription factor gene” refers to a gene thatencodes a protein that binds to regulatory regions and is involved incontrol gene expression. Therefore, as used herein, a target gene refersto a gene whose expression is controlled by a transcription factor gene.

As used herein, “percent identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, e.g., nucleotide sequence or aminoacid sequence. An “identity fraction” for aligned segments of a testsequence and a reference sequence is the number of identical componentswhich are shared by sequences of the two aligned segments divided by thetotal number of sequence components in the reference segment over awindow of alignment which is the smaller of the full test sequence orthe full reference sequence. “Percent identity” (“% identity”) is theidentity fraction times 100.

As used herein “Pfam” refers to a large collection of multiple sequencealignments and hidden Markov models covering many common proteinfamilies, e.g. Pfam version 19.0 (December 2005) contains alignments andmodels for 8183 protein families and is based on the Swissprot 47.0 andSP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “ProfileHidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam iscurrently maintained and updated by a Pfam Consortium. The alignmentsrepresent some evolutionary conserved structure that has implicationsfor the protein's function. Profile hidden Markov models (profile HMMs)built from the Pfam alignments are useful for automatically recognizingthat a new protein belongs to an existing protein family even if thehomology by alignment appears to be low. Once one DNA is identified asencoding a protein which imparts an enhanced trait when expressed intransgenic plants, other DNA encoding proteins in the same proteinfamily are identified by querying the amino acid sequence of proteinencoded by candidate DNA against the Hidden Markov Model whichcharacterizes the Pfam domain using HMMER software, a current version ofwhich is provided in the appended computer listing. Candidate proteinsmeeting the gathering cutoff for the alignment of a particular Pfam arein the protein family and have cognate DNA that is useful inconstructing recombinant DNA for the use in the plant cells of thisinvention. Hidden Markov Model databases for use with HMMER software inidentifying DNA expressing protein in a common Pfam for recombinant DNAin the plant cells of this invention are also included in the appendedcomputer listing. The HMMER software and Pfam databases are version 19.0and were used to identify known domains in the proteins corresponding toamino acid sequence of SEQ ID NO: 194 through SEQ ID NO: 386. All DNAencoding proteins that have scores higher than the gathering cutoffdisclosed in Table 11 by Pfam analysis disclosed herein can be used inrecombinant DNA of the plant cells of this invention, e.g. for selectingtransgenic plants having enhanced agronomic traits. The relevant Pfamsfor use in this invention, as more specifically disclosed below, areFAD_binding_(—)4, MtN3_slv, Homeobox, FAD_binding_(—)6, RWP-RK, PMEI,FAD_binding_(—)7, RRM_(—)1, Transaldolase, RNA_pol_L, WD40, U-box,Cyclin_N, Skp1, Redoxin, DZC, PBP, TPP_enzyme_M, CBFD_NFYB_HMF,TPP_enzyme_N, PFK, Caleosin, Iso_dh, Ribosomal_L18p, Metallophos,zf-A20, Ras, BBE, NAF, PLDc, DUF1242, Pkinase, C2, p450, Pyridoxal_deC,FBD, UPF0005, HEAT_PBS, GST_N, PEP-utilizers, Alpha-amylase,Amino_oxidase, SRF-TF, Phi_(—)1, Malic_M, Tryp_alpha_amyl, GSHPx, Miro,HSF-DNA-bind, DNA_photolyase, Sina, CTP_transf 2, Abhydrolase_(—)3,Chal_sti_synt_C, ACP_syn_III_C, ADH_zinc_N, CSD, Globin, GATase_(—)2,Amidohydro_(—)1, HLH, HALZ, Amidohydro_(—)3, Lactamase_B, HSP20, DAO,DUF296, AT_hook, AWPM-19, Dimerisation, Suc_Fer-like, Methyltransf_(—)2,Aminotran_(—)3, PHD, MMR_HSR₁, Aldo_ket_red, zf-AN1, malic, Fasciclin,UPF0057, DUF221, Pkinase_Tyr, DnaJ, Cofilin_ADF, Orn_Arg_deC_N,Skp1_POZ, Asn_synthase, K-box, LRR_(—)2, Ribosomal_L12, Ammonium_transp,Ribosomal_L14, KOW, DUF1336, DS, Aa_trans, CcmH, peroxidase, eIF-5a,Aldedh, PEP-utilizers_C, ADH_N, UIM, NAD_binding_(—)1, zf-C3HC4,Spermine_synth, AUX_IAA, LIM, Anti-silence, X8, Citrate_synt, 14-3-3,RMMBL, efhand, NPH3, CAF1, ICL, FAE1_CUT1_RppA, Orn_DAP_Arg_deC, PPDK_N,Myb_DNA-binding, AP2, F-box, and APS_kinase

As used herein, “promoter” means regulatory DNA for initializingtranscription. A “plant promoter” is a promoter capable of initiatingtranscription in plant cells whether or not its origin is a plant cell,e.g., is it well known that viral promoters are functional in plants.Thus, plant promoters include promoter DNA obtained from plants, plantviruses, and bacteria such as Agrobacterium and Rhizobium bacteria.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, or seeds. Such promoters are referred to as “tissuepreferred”. Promoters which initiate transcription only in certaintissues are referred to as “tissue specific”. A “cell type” specificpromoter primarily drives expression in certain cell types in one ormore organs, for example, vascular cells in roots or leaves. An“inducible” or “repressible” promoter is a promoter which is underenvironmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobicconditions, or certain chemicals, or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions.

As used herein, “operably linked” means the association of two or moreDNA fragments in a DNA construct so that the function of one, e.g.,protein-encoding DNA, is affected by the other, e.g., a promoter.

As used herein, “expression” means the process that includestranscription of DNA to produce RNA and translation of the cognateprotein encoded by the DNA and RNA.

As used herein, a “control plant” means a plant that does not containthe recombinant DNA that confers an enhanced agronomic trait. A controlplant is used to compare against a transgenic plant, to identify anenhanced agronomic trait in the transgenic plant. A suitable controlplant may be a non-transgenic plant of the parental line used togenerate a transgenic plant. A control plant may in some cases be atransgenic plant line that comprises an empty vector or marker gene, butdoes not contain the recombinant DNA.

As used herein, an “agronomic trait” means a characteristic of a plant,which includes, but are not limited to, plant morphology, physiology,growth and development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance. In the plants ofthis invention the expression of identified recombinant DNA confers anagronomically important trait, e.g., increased yield. An “enhancedagronomic trait” refers to a measurable improvement in an agronomictrait including, but not limited to, yield increase, including increasedyield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions may include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. “Yield” can be affected by many properties including withoutlimitation, plant height, pod number, pod position on the plant, numberof internodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Yield can also affected by efficiency ofgermination (including germination in stressed conditions), growth rate(including growth rate in stressed conditions), ear number, seed numberper ear, seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill.

Increased yield of a transgenic plant of the present invention can bemeasured in a number of ways, including test weight, seed number perplant, seed weight, seed number per unit area (i.e. seeds, or weight ofseeds, per acre), bushels per acre, tones per acre, tons per acre, kiloper hectare. For example, maize yield may be measured as production ofshelled corn kernels per unit of production area, e.g., in bushels peracre or metric tons per hectare, often reported on a moisture adjustedbasis, e.g., at 15.5% moisture. Increased yield may result from enhancedutilization of key biochemical compounds, such as nitrogen, phosphorousand carbohydrate, or from improved responses to environmental stresses,such as cold, heat, drought, salt, and attack by pests or pathogens.Recombinant DNA used in this invention can also be used to provideplants having enhanced growth and development, and ultimately increasedyield, as the result of modified expression of plant growth regulatorsor modification of cell cycle or photosynthesis pathways.

Also of interest is the generation of transgenic plants that demonstrateenhanced yield with respect to a seed component that may or may notcorrespond to an increase in overall plant yield. Such propertiesinclude enhancements in seed oil, seed molecules such as tocopherol,protein and starch, or oil particular oil components as may be manifestby an alteration in the ratios of seed components.

A subset of the nucleic molecules of this invention includes fragmentsof the disclosed recombinant DNA consisting of oligonucleotides of atleast 15, preferably at least 16 or 17, more preferably at least 18 or19, and even more preferably at least 20 or more, consecutivenucleotides. Such oligonucleotides are fragments of the larger moleculeshaving a sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:193, and find use, for example as probes and primersfor detection of the polynucleotides of the present invention.

In some embodiments of the invention a constitutively active mutant isconstructed to achieve the desired effect. SEQ ID NO: 3-6 encodes onlythe kinase domain of a calcium dependent protein kinase (CDPK). CDPK1has a domain structure similar to other calcium-dependant protein kinasein which the protein kinase domain is separated from four efhand domainsby 42 amino acid “spacer” region. Calcium-dependent protein kinases arethought to be activated by a calcium-induced conformational change thatresults in movement of an autoinhibitory domain away form the proteinkinase active site (Yokokura et al., 1995). Thus, consitutively activeproteins can be made by over expressing the protein kinase domain alone.

In other embodiments of the invention a chimeric gene is constructedbetween homologous genes from different species to obtain a protein withcertain characteristics superior to either native protein, e.g.,enhanced stability and favorable enzymatic kinetics. Exemplary chimericDNA molecules provided by the present invention are set forth as SEQ IDNO: 1 and 2 that encode a Arabidopsis-Corn chimeric pyruvateorthophosphate dikinase (PPDK).

In yet other embodiments of the invention, a codon optimized gene issynthesized to achieve a desirable expression level. Synthetic DNAmolecules can be designed by a variety of methods, such as, methodsknown in the art that are based upon substituting the codon(s) of afirst polynucleotide to create an equivalent, or even an improved,second-generation artificial polynucleotide, where this new artificialpolynucleotide is useful for enhanced expression in transgenic plants.The design aspect often employs a codon usage table. The table isproduced by compiling the frequency of occurrence of codons in acollection of coding sequences isolated from a plant, plant type, familyor genus. Other design aspects include reducing the occurrence ofpolyadenylation signals, intron splice sites, or long AT or GC stretchesof sequence (U.S. Pat. No. 5,500,365). Full length coding sequences orfragments thereof can be made of artificial DNA using methods known tothose skilled in the art. Such exemplary synthetic DNA moleculesprovided by the present invention are set forth as SEQ ID NO: 38.

DNA constructs are assembled using methods well known to persons ofordinary skill in the art and typically comprise a promoter operablylinked to DNA, the expression of which provides the enhanced agronomictrait. Other construct components may include additional regulatoryelements, such as 5′ introns for enhancing transcription, 3′untranslated regions (such as polyadenylation signals and sites), DNAfor transit or signal peptides.

In accordance with the current invention, constitutive promoters areactive under most environmental conditions and states of development orcell differentiation. These promoters are likely to provide expressionof the polynucleotide sequence at many stages of plant development andin a majority of tissues. A variety of constitutive promoters are knownin the art. Examples of constitutive promoters that are active in plantcells include but are not limited to the nopaline synthase (NOS)promoters; the cauliflower mosaic virus (CaMV) 19S and 35S promoters(U.S. Pat. No. 5,858,642); the figwort mosaic virus promoter (P-FMV,U.S. Pat. No. 6,051,753); actin promoters, such as the rice actinpromoter (P-Os.Act1, U.S. Pat. No. 5,641,876).

Furthermore, the promoters may be altered to contain one or more“enhancer sequences” to assist in elevating gene expression. Suchenhancers are known in the art. By including an enhancer sequence withsuch constructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedin the forward or reverse orientation 5′ or 3′ to the coding sequence.In some instances, these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1(see U.S. Pat. No. 5,641,876), rice actin 2 genes and the maize heatshock protein 70 gene intron (U.S. Pat. No. 5,593,874). Examples ofother enhancers that can be used in accordance with the inventioninclude elements from the CaMV 35S promoter, octopine synthase genes,the maize alcohol dehydrogenase gene, the maize shrunken 1 gene andpromoters from non-plant eukaryotes.

Tissue-specific promoters cause transcription or enhanced transcriptionof a polynucleotide sequence in specific cells or tissues at specifictimes during plant development, such as in vegetative or reproductivetissues. Examples of tissue-specific promoters under developmentalcontrol include promoters that initiate transcription primarily incertain tissues, such as vegetative tissues, e.g., roots, leaves orstems, or reproductive tissues, such as fruit, ovules, seeds, pollen,pistils, flowers, or any embryonic tissue, or any combination thereof.Reproductive tissue specific promoters may be, e.g., ovule-specific,embryo-specific, endosperm-specific, integument-specific,pollen-specific, petal-specific, sepal-specific, or some combinationthereof. Tissue specific promoter(s) will also include promoters thatcan cause transcription, or enhanced transcription in a desired planttissue at a desired plant developmental stage. An example of such apromoter includes, but is not limited to, a seedling or an earlyseedling specific promoter. One skilled in the art will recognize that atissue-specific promoter may drive expression of operably linkedpolynucleotide molecules in tissues other than the target tissue. Thus,as used herein, a tissue-specific promoter is one that drives expressionpreferentially not only in the target tissue, but may also lead to someexpression in other tissues as well.

In one embodiment of this invention, preferential expression in plantgreen tissues is desired. Promoters of interest for such uses includethose from genes such as maize aldolase gene FDA (U.S. patentapplication publication No. 20040216189), aldolase and pyruvateorthophosphate dikinase (PPDK) (Taniguchi et al. (2000) Plant CellPhysiol. 41(1):42-48).

In another embodiment of this invention, preferential expression inplant root tissue is desired. An exemplary promoter of interest for suchuses is derived from Corn Nicotianamine Synthase gene (U.S. patentapplication publication No. 20030131377).

In yet another embodiment of this invention, preferential expression inplant phloem tissue is desired. An exemplary promoter of interest forsuch use is the rice tungro bacilliform virus (RTBV) promoter (U.S. Pat.No. 5,824,857).

In practicing this invention, an inducible promoter may also be used toectopically express the structural gene in the recombinant DNAconstruct. The inducible promoter may cause conditional expression of apolynucleotide sequence under the influence of changing environmentalconditions or developmental conditions. For example, such promoters maycause expression of the polynucleotide sequence at certain temperaturesor temperature ranges, or in specific stage(s) of plant development suchas in early germination or late maturation stage(s) of a plant. Examplesof inducible promoters include, but are not limited to, thelight-inducible promoter from the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Fischhoff et al.(1992) Plant Mol. Biol. 20:81-93); the drought-inducible promoter ofmaize (Busk et al., Plant J. 11:1285-1295, 1997), the cold, drought, andhigh salt inducible promoter from potato (Kirch, Plant Mol. Biol.33:897-909, 1997), and many cold inducible promoters known in the art;for example rd29a and cor15a promoters from Arabidopsis (Genbank ID:D13044 and U01377), blt101 and blt4.8 from barley (Genbank ID: AJ310994and U63993), wcs 120 from wheat (Genbank ID:AF031235), mlip15 from corn(Genbank ID: D26563) and bn115 from Brassica (Genbank ID: U01377).

In some aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2):157-166), glutelin1 (Russell (1997) supra),peroxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol. Biol.31(6):1205-1216), and globulin 1 (Belanger et al (1991) Genetics129:863-872).

Recombinant DNA constructs prepared in accordance with the inventionwill also generally include a 3′ element that typically contains apolyadenylation signal and site. Well-known 3′ elements include thosefrom Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms3′, ocs 3′, tr7 3′, e.g., disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference; 3′ elements from plant genes such aswheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheatubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelingene a rice lactate dehydrogenase gene and a rice beta-tubulin gene, allof which are disclosed in U.S. published patent application 2002/0192813A1, incorporated herein by reference; and the pea (Pisum sativum)ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from thegenes within the host plant.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925, incorporated herein by reference. For description of thetransit peptide region of an Arabidopsis EPSPS gene useful in thepresent invention, see Klee, H. J. et al., (MGG (1987) 210:437-442).

The recombinant DNA construct may include other elements. For example,the construct may contain DNA segments that provide replication functionand antibiotic selection in bacterial cells. For example, the constructmay contain an E. coli origin of replication such as ori322 or a broadhost range origin of replication such as oriV, oriRi or oriColE.

The construct may also comprise a selectable marker such as anEc-ntpII-Tn5 that encodes a neomycin phosphotransferase II gene obtainedfrom Tn5 conferring resistance to a neomycin and kanamysin, Spc/Str thatencodes for Tn7 aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)or one of many known selectable marker gene.

The vector or construct may also include a screenable marker and otherelements as appropriate for selection of plant or bacterial cells havingDNA constructs of the invention. DNA constructs are designed withsuitable selectable markers that can confer antibiotic or herbicidetolerance to the cell. The antibiotic tolerance polynucleotide sequencesinclude, but are not limited to, polynucleotide sequences encoding forproteins involved in tolerance to kanamycin, neomycin, hygromycin, andother antibiotics known in the art. An antibiotic tolerance gene in sucha vector may be replaced by herbicide tolerance gene encoding for5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S.Pat. Nos. 5,627,061, and 5,633,435; Padgette et al., Herbicide ResistantCrops, Lewis Publishers, 53-85, 1996; and in Penaloza-Vazquez, et al.,Plant Cell Reports 14:482-487, 1995) and aroA (U.S. Pat. No. 5,094,945)for glyphosate tolerance, bromoxynil nitrilase (Bxn) for Bromoxyniltolerance (U.S. Pat. No. 4,810,648), phytoene desaturase (crtI (Misawaet al., Plant J. 4:833-840, 1993; and Misawa et al., Plant J. 6:481-489,1994) for tolerance to norflurazon, acetohydroxyacid synthase (AHAS,Sathasiivan et al., Nucl. Acids Res. 18:2188-2193, 1990). Herbicides forwhich transgenic plant tolerance has been demonstrated and for which themethod of the present invention can be applied include, but are notlimited to: glyphosate, sulfonylureas, imidazolinones, bromoxynil,delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, andisoxaslutole herbicides.

Other examples of selectable markers, screenable markers and otherelements are well known in the art and may be readily used in thepresent invention. Those skilled in the art should refer to thefollowing for details (for selectable markers, see Potrykus et al., Mol.Gen. Genet. 199:183-188, 1985; Hinchee et al., Bio. Techno. 6:915-922,1988; Stalker et al., J. Biol. Chem. 263:6310-6314, 1988; EuropeanPatent Application 154,204; Thillet et al., J. Biol. Chem.263:12500-12508, 1988; for screenable markers see, Jefferson, Plant Mol.Biol, Rep. 5: 387-405, 1987; Jefferson et al., EMBO J. 6: 3901-3907,1987; Sutcliffe et al., Proc. Natl. Acad. Sci. U.S.A. 75: 3737-3741,1978; Ow et al., Science 234: 856-859, 1986; Ikatu et al., Bio. Technol.8: 241-242, 1990; and for other elements see, European PatentApplication Publication Number 0218571; Koziel et al., Plant Mol. Biol.32: 393-405; 1996).

The plants of this invention can be further enhanced with stackedtraits, e.g., a crop having an enhanced agronomic trait resulting fromexpression of DNA disclosed herein, in combination with herbicide,disease, and/or pest resistance traits. The recombinant DNA is providedin plant cells derived from corn lines that maintain resistance to avirus such as the Mal de Rio Cuarto virus or a fungus such as thePuccina sorghi fungus or both, which are common plant diseases inArgentina. For example, genes of the current invention can be stackedwith other traits of agronomic interest, such as a trait providingherbicide resistance, or insect resistance, such as using a gene fromBacillus thuringiensis to provide resistance against lepidopteran,coliopteran, homopteran, hemiopteran, and other insects. Herbicides forwhich transgenic plant tolerance has been demonstrated and the method ofthe present invention can be applied include, but are not limited to,glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil andnorflurazon herbicides. Polynucleotide molecules encoding proteinsinvolved in herbicide tolerance are well-known in the art and include,but are not limited to, a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S.Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for impartingglyphosate tolerance; polynucleotide molecules encoding a glyphosateoxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and aglyphosate-N-acetyl transferase (GAT) disclosed in U.S. PatentApplication publication 2003/0083480 A1 also for imparting glyphosatetolerance; dicamba monooxygenase disclosed in U.S. Patent Applicationpublication 2003/0135879 A1 for imparting dicamba tolerance; apolynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed inU.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; apolynucleotide molecule encoding phytoene desaturase (crtI) described inMisawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) PlantJ. 6:481-489 for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for impartingtolerance to sulfonylurea herbicides; polynucleotide molecules known asbar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 forimparting glufosinate and bialaphos tolerance; polynucleotide moleculesdisclosed in U.S. Patent Application Publication 2003/010609 A1 forimparting N-amino methyl phosphonic acid tolerance; polynucleotidemolecules disclosed in U.S. Pat. No. 6,107,549 for impartinig pyridineherbicide resistance; molecules and methods for imparting tolerance tomultiple herbicides such as glyphosate, atrazine, ALS inhibitors,isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No.6,376,754 and U.S. Patent Application Publication 2002/0112260, all ofsaid U.S. patents and Patent Application Publications are incorporatedherein by reference. Molecules and methods for impartinginsect/nematode/virus resistance is disclosed in U.S. Pat. Nos.5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent ApplicationPublication 2003/0150017 A1, all of which are incorporated herein byreference.

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the proteinsdisclosed herein. Means for preparing and characterizing antibodies arewell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include using glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified antifungal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp.75-83). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag-4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Spend virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserineAminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azasenne blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. Nos. 5,015,580(soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean);6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) andAgrobacterium-mediated transformation is described in U.S. Pat. Nos.5,159,135 (cotton); 5,824,877 (soybean); 5,591,616 (corn); and 6,384,301(soybean), and in US Patent Application Publication 2004/0244075, all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, e.g., to replace an existing gene inthe genome, to use an existing promoter in the plant genome, or toinsert a recombinant polynucleotide at a predetermined site known to beactive for gene expression. Several site specific recombination systemsexist which are known to function implants include cre-lox as disclosedin U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, e.g., various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for screening of plantshaving an enhanced agronomic trait. In addition to direct transformationof a plant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant which can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced agronomic trait, e.g.,enhanced yield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, e.g., herbicide resistanceor pest resistance, to produce progeny plants having recombinant DNAthat confers both traits. Typically, in such breeding for combiningtraits the transgenic plant donating the additional trait is a male lineand the transgenic plant carrying the base traits is the female line.The progeny of this cross will segregate such that some of the plantswill carry the DNA for both parental traits and some will carry DNA forone parental trait; such plants can be identified by markers associatedwith parental recombinant DNA Progeny plants carrying DNA for bothparental traits can be crossed back into the female parent line multipletimes, e.g., usually 6 to 8 generations, to produce a progeny plant withsubstantially the same genotype as one original transgenic parental linebut for the recombinant DNA of the other transgenic parental line.

In the practice of transformation DNA is typically introduced into onlya small percentage of target cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga transgenic DNA construct into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or herbicide. Any of the herbicides to whichplants of this invention may be resistant are useful agents forselective markers. Potentially transformed cells are exposed to theselective agent. In the population of surviving cells will be thosecells where, generally, the resistance-conferring gene is integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.Commonly used selective marker genes include those conferring resistanceto antibiotics such as kanamycin and paromomycin (nptII), hygromycin B(aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicidessuch as glufosinate (bar or pat) and glyphosate (aroA or EPSPS).Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047, all of which are incorporated hereinby reference. Screenable markers which provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets can be transferred to plant growth mix, and hardened off,e.g., in an environmentally controlled chamber at about 85% relativehumidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, priorto transfer to a greenhouse or growth chamber for maturation. Plants areregenerated from about 6 weeks to 10 months after a transformant isidentified, depending on the initial tissue. Plants may be pollinatedusing conventional plant breeding methods known to those of skill in theart and seed produced, e.g., self-pollination is commonly used withtransgenic corn. The regenerated transformed plant or its progeny seedor plants can be tested for expression of the recombinant DNA andscreened for the presence of enhanced agronomic trait.

Transgenic plant seed provided by this invention are grown to generatetransgenic plants having an enhanced trait as compared to a controlplant. Such seed for plants with enhanced agronomic trait is identifiedby screening transformed plants or progeny seed for enhanced trait. Forefficiency a screening program is designed to evaluate multipletransgenic plants (events) comprising the recombinant DNA, e.g.,multiple plants from 2 to 20 or more transgenic events.

Transgenic plants grown from transgenic seed provided herein demonstrateenhanced agronomic traits that contribute to increased yield or othertrait that provides increased plant value, including, for example,enhanced seed quality. Of particular interest are plants having enhancedyield resulting from enhanced plant growth and development, stresstolerance, enhanced seed development, higher light response, enhancedflower development, or enhanced carbon and/or nitrogen metabolism.

Table 1 provides a list of protein encoding DNA (“genes”) that areuseful as recombinant DNA for production of transgenic plants withenhanced agronomic trait, the elements of Table 1 are described byreference to:

“NUC SEQ ID NO” which is a SEQ ID NO for a DNA sequence in the SequenceListing.“PEP SEQ ID NO” which is a SEQ ID NO for an amino acid sequence in theSequence Listing.GENE ID″ which is an arbitrary name for the recombinant DNA.“Base Vector” which is a reference to the identifying number in Table 5of base vectors used for transformation of the recombinant DNA.Construction of plant transformation constructs is illustrated inExample 1.“annotation” refers to a description of the top hit protein obtainedfrom an amino acid sequence query of each PEP SEQ ID NO to GenBankdatabase of the National Center for Biotechnology Information (NCBI).Identifier is the GenBank ID number for the informative BLAST hit with—F T.

TABLE 1 NUC PEP annotation SEQ SEQ Base e- % ID NO ID NO vector GENE IDvalue identity identifier description 1 194 1 PHE0003351_PMON81242 0 98168586 gb|AAA33498.1|pyruvate, orthophosphate dikinase 2 195 7PHE0003351_PMON83625 0 98 168586 gb|AAA33498.1|pyruvate, orthophosphatedikinase 3 196 1 PHE0000207_PMON77878 1.00E−144 96 34907990ref|NP_915342.1|putative calcium-dependent protein kinase [Oryza sativa(japonica cultivar-group)] 4 197 1 PHE0000208_PMON77879 1.00E−143 9450919297 ref|XP_470045.1|putative calmodulin-domain protein kinase[Oryza sativa (japonica cultivar-group)] 5 198 1 PHE0000209_PMON778911.00E−135 89 53850561 gb|AAU95457.1|At5g12180 [Arabidopsis thaliana]dbj|BAB10036.1|calcium- dependent protein kinase 6 199 1PHE0000210_PMON77880 1.00E−137 89 26452430 dbj|BAC43300.1|putativecalcium-dependent protein kinase [Arabidopsis thaliana] 7 200 8PHE0001329_PMON92878 0 100 34903780 dbj|BAB92151.1|putativeCBL-interacting protein kinase 2 [Oryza sativa (japonica 8 201 1PHE0001425_PMON79162 1.00E−154 100 51979679 ref|XP_507586.1|PREDICTEDP0524F03.33 gene product [Oryza sativa (japonica cultivar-group)]ref|XP_482612.1|putative CCR4-NOT transcription complex, subunit 7 9 2028 PHE0001573_PMON92870 0 78 984262 emb|CAA58052.1|asparragine synthetase[Zea mays] 10 203 12 PHE0001664_PMON99280 0 100 34906358sp|Q9LDE6|CKX1_ORYSA Probable cytokinin dehydrogenase precursor(Cytokinin oxidase) (CKO) 11 204 1 PHE0001674_PMON79194 5.00E−12 5015223390 ref|NP_171645.1|myb family transcription factor [Arabidopsisthaliana] 12 205 10 PHE0002026_PMON96489 0 87 32488298emb|CAE03364.1|OSJNBb0065L13.7 [Oryza sativa (japonica cultivar-group)]13 206 8 PHE0002108_PMON92821 2.00E−31 100 10176234dbj|BAB07329.1|cold-shock protein [Bacillus halodurans C-125] 14 207 8PHE0002109_PMON93856 6.00E−33 100 41324401 emb|CAF18741.1|COLD- SHOCKPROTEIN CSPA [Corynebacterium glutamicum ATCC 13032] 15 208 8PHE0002508_PMON92607 2.00E−79 72 50509850 dbj|BAD32022.1|putativetranscription factor [Oryza sativa 16 209 1 PHE0002650_PMON818321.00E−132 100 9964296 gb|AAG09919.1|MADS box protein 2 [Zea mays] 17 2102 PHE0002989_PMON95630 1.00E−117 100 7271044 emb|CAB80652.1|smallGTP-binding protein-like [Arabidopsis thaliana] 18 211 6PHE0003290_PMON95107 4.00E−29 34 7269078 emb|CAB79187.1|hypotheticalprotein [Arabidopsis thaliana] 19 212 6 PHE0003300_PMON95106 7.00E−81 5450908933 ref|XP_465955.1|putative nodulin 3 [Oryza sativa (japonicacultivar-group)] 20 213 6 PHE0003303_PMON95080 2.00E−96 69 38347194emb|CAD37109.2|OSJNBa0024J22.22 [Oryza sativa (japonica cultivar-group)]21 214 8 PHE0003389_PMON94682 0 65 52076827 dbj|BAD45770.1|putativeCyt-P450 monooxygenase [Oryza sativa (japonica cultivar-group)] 22 215 8PHE0003614_PMON95111 0 94 32309578 gb|AAP79441.1|glutamate decarboxylase[Oryza sativa (japonica cultivar-group)] 23 216 8 PHE0003684_PMON928071.00E−72 68 34906004 dbj|BAB63676.1|induced protein MgI1 [Oryza sativa(japonica cultivar-group)] 24 217 9 PHE0003684_PMON93378 1.00E−72 6834906004 dbj|BAB63676.1|induced protein MgI1 [Oryza sativa (japonicacultivar-group)] 25 218 8 PHE0003853_PMON92602 1.00E−179 98 62320210ref|NP_195478.2|cyclin family protein [Arabidopsis thaliana]gb|AAS49095.1| At4g37630 [Arabidopsis thaliana] 26 219 11PHE0003903_PMON98271 0 99 19851522 gb|AAL99744.1|pyruvate decarboxylase[Zea mays] 27 220 11 PHE0003905_PMON99283 0 92 11995457gb|AAG43027.1|aldehyde dehydrogenase [Oryza sativa] 28 221 11PHE0003907_PMON98066 5.00E−87 86 50906015 ref|XP_464496.1|ribosomalprotein L12-like protein [Oryza sativa (japonica cultivar-group)] 29 22211 PHE0003908_PMON98064 0 84 51535811 dbj|BAD37896.1|ARG1- like protein[Oryza sativa (japonica cultivar-group)] 30 223 6 PHE0003960_PMON950791.00E−156 87 50905641 ref|XP_464309.1|putative choline-phosphatecytidylyltransferase [Oryza sativa (japonica cultivar- group)] 31 224 5PHE0003967_PMON95088 1.00E−102 83 55168334gb|AAV44199.1|dehydroascorbate reductase [Oryza sativa (japonicacultivar- group)] 32 225 10 PHE0003985_PMON96457 1.00E−30 58 55770043ref|XP_550011.1|hypothetical protein [Oryza sativa (japonicacultivar-group)] 33 226 10 PHE0003987_PMON96134 5.00E−41 74 50919885ref|XP_470303.1|hypothetical protein [Oryza sativa (japonicacultivar-group)] 34 227 10 PHE0004001_PMON96453 4.00E−22 66 51978970ref|XP_507362.1|PREDICTED OSJNBa0077F02.127 gene product [Oryza sativa(japonica cultivar-group)] 35 228 8 PHE0004023_PMON92446 1.00E−132 8812651665 gb|AAA20093.2|Alfin-1 [Medicago sativa] pir||T09646 probablezinc finger protein - alfalfa (fragment) 36 229 4 PHE0004026_PMON93885 0100 21592703 gb|AAM64652.1|LAX1/ AUX1-like permease [Arabidopsisthaliana] 37 230 4 PHE0004027_PMON93860 0 100 7269873emb|CAB79732.1|cytokinin oxidase-like protein [Arabidopsis thaliana] 38231 15 PHE0004028_PMON94697 0 100 216765 dbj|BAA14344.1|sucrosephosphorylase [Leuconostoc mesenteroides] 12607 231 n/aPHE0010424_PMON17730 0 100 216765 dbj|BAA14344.1|sucrose phosphorylase[Leuconostoc mesenteroides] 39 232 8 PHE0004034_PMON92631 0 100 6520233dbj|BAA87958.1|CW14 [Arabidopsis thaliana] 40 233 8 PHE0004039_PMON926341.00E−178 65 26452061 ref|NP_191207.2|myosin heavy chain-related[Arabidopsis thaliana] 41 234 8 PHE0004047_PMON92619 4.00E−79 7462087121 dbj|BAD91881.1|transcription factor lim1 [Eucalyptuscamaldulensis] 42 235 14 PHE0004047_PMON93388 4.00E−79 74 62087121dbj|BAD91881.1|transcription factor lim1 [Eucalyptus camaldulensis] 43236 8 PHE0004068_PMON93663 3.00E−94 100 15293293 ref|NP_563710.1|AWPM-19-like membrane family protein [Arabidopsis thaliana] 44 237 8PHE0004071_PMON93311 1.00E−130 100 21358850 ref|NP_568751.1|polyadenylate-binding protein, putative/PABP, putative [Arabidopsisthaliana] 45 238 8 PHE0004072_PMON93654 0 100 23297397ref|NP_192188.2|GTP- binding family protein [Arabidopsis thaliana] 46239 14 PHE0004072_PMON93669 0 100 23297397 ref|NP_192188.2|GTP- bindingfamily protein [Arabidopsis thaliana] 47 240 8 PHE0004074_PMON94164 0100 9759255 ref|NP_196133.3| transcription elongation factor-related[Arabidopsis thaliana] 48 241 8 PHE0004075_PMON92851 1.00E−132 10011994587 ref|NP_566493.1|nodulin MtN3 family protein [Arabidopsisthaliana] 49 242 8 PHE0004080_PMON93321 1.00E−143 99 16173emb|CAA42168.1|L- ascorbate peroxidase [Arabidopsis thaliana] 50 243 14PHE0004084_PMON95141 0 100 7267537 emb|CAB78019.1|putative phi-1-likephosphate- induced protein [Arabidopsis thaliana] gb|AAM18526.1|cellcycle- related protein [Arabidopsis thaliana] 51 244 8PHE0004093_PMON93332 0 100 12744973 gb|AAK06866.1|putative ATPase[Arabidopsis thaliana] ref|NP_173536.1| O-methyltransferase, putative[Arabidopsis thaliana] 52 245 14 PHE0004093_PMON94155 0 100 12744973gb|AAK06866.1|putative ATPase [Arabidopsis thaliana] ref|NP_173536.1|O-methyltransferase, putative [Arabidopsis thaliana] 53 246 8PHE0004139_PMON92898 2.00E−88 100 21554099 ref|NP_568761.1|expressedprotein [Arabidopsis thaliana] 54 247 8 PHE0004144_PMON93842 1.00E−78100 21555039 ref|NP_565390.1|actin- depolymerizing factor 5 (ADF5)[Arabidopsis thaliana] 55 248 8 PHE0004148_PMON92574 0 100 48768596ref|ZP_00272945.1|COG0538: Isocitrate dehydrogenases [Ralstoniametallidurans CH34] 56 249 8 PHE0004149_PMON92471 1.00E−148 99 31096331ref|NP_441003.1| phycocyanin alpha phycocyanobilin lyase; CpcE[Synechocystis sp. PCC 6803] 57 250 14 PHE0004149_PMON93899 1.00E−148 9931096331 ref|NP_441003.1| phycocyanin alpha phycocyanobilin lyase; CpcE[Synechocystis sp. PCC 6803] 58 251 15 PHE0004152_PMON93672 3.00E−85 608978267 ref|NP_199781.1|DNA- binding protein-related [Arabidopsisthaliana] 59 252 8 PHE0004155_PMON92626 0 100 22136876ref|NP_200010.1|sorbitol dehydrogenase, putative/ L-iditol2-dehydrogenase, putative [Arabidopsis thaliana] 60 253 8PHE0004156_PMON92623 0 98 12322729 ref|NP_187478.1|phototropic-responsive protein, putative [Arabidopsis thaliana] 61 254 8PHE0004162_PMON92481 3.00E−77 57 7269806 emb|CAB79666.1|phytochrome-associated protein PAP2 [Arabidopsis thaliana] 62 255 8PHE0004164_PMON92465 4.00E−67 100 21537028 ref|NP_198423.1|glycosylhydrolase family protein 17 [Arabidopsis thaliana] 63 256 8PHE0004166_PMON93801 6.00E−09 100 13374861 emb|CAC34495.1|putativestrictosidine synthase-like [Arabidopsis thaliana] 64 257 8PHE0004167_PMON93333 1.00E−176 100 28827764 ref|NP_569050.1|adenylylsulfate kinase, putative [Arabidopsis thaliana] 65 258 8PHE0004168_PMON93855 0 100 18176302 ref|NP_199253.1|FAD- bindingdomain-containing protein [Arabidopsis thaliana] 66 259 8PHE0004169_PMON92568 0 100 5080826 gb|AAD39335.1|Putative Aldo/ketoreductase [Arabidopsis thaliana] 67 260 8 PHE0004184_PMON92565 0 1007270846 emb|CAB80527.1|multiubiquitin chain binding protein (MBP1)[Arabidopsis thaliana] 68 261 8 PHE0004185_PMON92802 0 100 28460683ref|NP_182075.1| cytochrome P450, putative [Arabidopsis thaliana] 69 2628 PHE0004188_PMON92803 0 100 20465485 ref|NP_200218.1|heat shocktranscription factor family protein [Arabidopsis thaliana] 70 263 8PHE0004190_PMON92801 1.00E−167 98 7267277 ref|NP_192426.1|basichelix-loop-helix (bHLH) family protein [Arabidopsis thaliana] 71 264 8PHE0004208_PMON92834 1.00E−83 55 21555865 gb|AAS09998.1|MYBtranscription factor [Arabidopsis thaliana] 72 265 8PHE0004215_PMON92827 2.00E−55 65 7320708 ref|NP_195750.1|phosphatidylethanolamine- binding family protein [Arabidopsis thaliana]73 266 8 PHE0004223_PMON92840 0 100 6523058 ref|NP_190239.1|fasciclin-like arabinogalactan family protein [Arabidopsis thaliana] 74 267 8PHE0004225_PMON94167 0 99 1421730 gb|AAC49371.1|RF2 gb|AAG43988.1|Tcytoplasm male sterility restorer factor 2 [Zea mays] 75 268 10PHE0004226_PMON95114 0 100 53793208 dbj|BAD54414.1|aldehydedehydrogenase ALDH2b [Oryza sativa (japonica cultivar-group)] 76 269 8PHE0004227_PMON92605 5.00E−26 100 21314334 gb|AAM46894.1|early droughtinduced protein [Oryza sativa (indica cultivar-group)] 77 270 8PHE0004229_PMON92867 1.00E−24 100 6320482 ref|NP_010562.1|Small plasmamembrane protein related to a family of plant polypeptides that areoverexpressed under high salt concentration or low temperature, notessential for viability, deletion causes hyperpolarization of the plasmamembrane potential; Pmp3p [Saccharomyces cerevisiae] 78 271 8PHE0004233_PMON92843 0 100 19310749 ref|NP_188922.1|heat shocktranscription factor family protein [Arabidopsis thaliana] 79 272 13PHE0004237_PMON93673 9.00E−85 100 16338 emb|CAA45039.1|heat shockprotein 17.6-II [Arabidopsis thaliana] 80 273 8 PHE0004243_PMON926213.00E−72 82 30409461 dbj|BAC76332.1|HAP3 [Oryza sativa (japonicacultivar-group)] 81 274 8 PHE0004244_PMON92858 1.00E−159 96 15321716gb|AAK95562.1|leafy cotyledon1 [Zea mays] 82 275 8 PHE0004245_PMON938131.00E−131 100 50509850 dbj|BAD32022.1|putative transcription factor[Oryza sativa (japonica cultivar- group)] 83 276 8 PHE0004248_PMON946721.00E−98 100 34907184 ref|NP_914939.1|putative CCAAT-bindingtranscription factor subunit A(CBF-A) [Oryza sativa 84 277 8PHE0004249_PMON95137 1.00E−48 100 12642910 ref|NP_850005.1|expressedprotein [Arabidopsis thaliana] 85 278 8 PHE0004250_PMON92881 5.00E−78100 30409463 dbj|BAC76333.1|HAP3 [Oryza sativa (japonicacultivar-group)] 86 279 8 PHE0004252_PMON92606 1.00E−173 100 18481620gb|AAL73485.1|repressor protein [Oryza sativa] 87 280 8PHE0004253_PMON92874 1.00E−143 100 18481626 gb|AAL73488.1|repressorprotein [Zea mays] 88 281 14 PHE0004258_PMON93385 0 100 1871189gb|AAB63549.1|putative protein kinase [Arabidopsis thaliana] 89 282 8PHE0004258_PMON93806 0 100 1871189 gb|AAB63549.1|putative protein kinase[Arabidopsis thaliana] 90 283 14 PHE0004259_PMON93384 0 100 9755654ref|NP_197112.1|expressed protein [Arabidopsis thaliana] 91 284 8PHE0004260_PMON92854 1.00E−48 100 12642910 ref|NP_850005.1|expressedprotein [Arabidopsis thaliana] 92 285 14 PHE0004261_PMON93389 1.00E−170100 7270230 ref|NP_195009.1|protein kinase, putative [Arabidopsisthaliana] 93 286 8 PHE0004261_PMON93655 1.00E−170 100 7270230ref|NP_195009.1|protein kinase, putative [Arabidopsis thaliana] 94 287 8PHE0004262_PMON92862 0 100 42570809 ref|NP_973478.1|protein kinase,putative [Arabidopsis thaliana] 95 288 14 PHE0004262_PMON93360 0 10042570809 ref|NP_973478.1|protein kinase, putative [Arabidopsis thaliana]96 289 8 PHE0004264_PMON92845 3.00E−95 100 21554624 ref|NP_201267.1|invertase/pectin methylesterase inhibitor family protein [Arabidopsisthaliana] 97 290 14 PHE0004264_PMON93354 3.00E−95 100 21554624ref|NP_201267.1| invertase/pectin methylesterase inhibitor familyprotein [Arabidopsis thaliana] 98 291 8 PHE0004265_PMON92873 0 100642305 ref|NP_013662.1| Hypothetical ORF; Yml050wp [Saccharomycescerevisiae] 99 292 14 PHE0004265_PMON93807 0 100 642305 ref|NP_013662.1|Hypothetical ORF; Yml050wp [Saccharomyces cerevisiae] 100 293 8PHE0004266_PMON92877 0 99 23506085 ref|NP_567548.1|pseudo- responseregulator 2 (APRR2) (TOC2) [Arabidopsis thaliana] 101 294 8PHE0004284_PMON93857 0 99 18399375 ref|NP_566402.1|U-boxdomain-containing protein [Arabidopsis thaliana] 102 295 10PHE0004285_PMON95136 1.00E−161 96 37542675 gb|AAL47207.1|HAP3-liketranscriptional-activator [Oryza sativa (indica cultivar-group)] 103 2968 PHE0004286_PMON93666 0 99 255220 gb|AAB23208.1|isocitrate lyase,threo-D S-isocitrate glyoxylate-lyase, IL {EC 4.1.3.1} [Brassica napus,seedlings, Peptide, 576 aa] 104 297 8 PHE0004287_PMON93344 0 88 50937953ref|XP_478504.1|putative isocitrate lyase [Oryza sativa (japonicacultivar- group)] 105 298 2 PHE0004307_PMON94102 1.00E−105 62 38345397emb|CAE03088.2|OSJNBa0017B10.3 [Oryza sativa (japonica cultivar-group)]106 299 14 PHE0004314_PMON93397 9.00E−52 54 55740645gb|AAV63915.1|hypothetical protein At4g03965 [Arabidopsis thaliana] 107300 8 PHE0004321_PMON93811 1.00E−128 100 18655355 sp|O48646|GPX4_ARATHProbable phospholipid hydroperoxide glutathione peroxidase,mitochondrial precursor (PHGPx) (AtGPX1) 108 301 14 PHE0004321_PMON938341.00E−128 100 18655355 ref|NP_192897.2| glutathione peroxidase, putative[Arabidopsis thaliana] 109 302 8 PHE0004325_PMON93818 5.00E−78 8950906887 ref|XP_464932.1|cytochrome c biogenesis protein-like [Oryzasativa (japonica cultivar-group)] 110 303 8 PHE0004335_PMON93850 0 10028393953 gb|AAO42384.1|putative major intrinsic protein [Arabidopsisthaliana] 111 304 8 PHE0004336_PMON93858 1.00E−146 69 51964952ref|XP_482812.1|major intrinsic protein-like [Oryza sativa (japonicacultivar- group)] 112 305 4 PHE0004337_PMON93886 0 62 50943587ref|XP_481321.1|unknown protein [Oryza sativa (japonica cultivar-group)]113 306 8 PHE0004348_PMON93810 1.00E−32 100 15644431ref|NP_229483.1|cold shock protein [Thermotoga maritima MSB8] 114 307 8PHE0004349_PMON93812 8.00E−33 100 15644617 ref|NP_229670.1|cold shockprotein [Thermotoga maritima MSB8] 115 308 8 PHE0004350_PMON938263.00E−31 100 20808157 ref|NP_623328.1|Cold shock proteins[Thermoanaerobacter tengcongensis MB4] 116 309 8 PHE0004351_PMON938217.00E−32 100 56419891 ref|YP_147209.1|cold shock protein [Geobacilluskaustophilus HTA426] 117 310 8 PHE0004352_PMON93824 1.00E−27 88 49611845ref|YP_050486.1|cold shock protein [Erwinia carotovora subsp.atroseptica SCRI1043] 118 311 8 PHE0004383_PMON93816 1.00E−34 9850899510 ref|XP_450543.1|unknown protein [Oryza sativa (japonicacultivar-group)] 119 312 8 PHE0004393_PMON94192 8.00E−95 100 42572939ref|NP_974566.1|calcineurin B-like protein 1 (CBL1) [Arabidopsisthaliana] 120 313 8 PHE0004395_PMON94145 0 100 30690488ref|NP_849501.1|phospholipase D delta/PLD delta (PLDDELTA) [Arabidopsisthaliana] 121 314 8 PHE0004396_PMON94137 0 100 7270422emb|CAB80188.1|arginine decarboxylase SPE2 [Arabidopsis thaliana] 122315 8 PHE0004417_PMON94190 1.00E−170 100 1230677 gb|AAC17191.1|spermidine synthase [Saccharomyces cerevisiae] 123 316 8PHE0004418_PMON94368 0 100 798930 sp|P50264|FMS1_YEAST Polyamine oxidaseFMS1 (Fenpropimorph resistance multicopy suppressor 1) 124 317 8PHE0004419_PMON95100 0 66 21281139 ref|NP_567276.1| amidohydrolasefamily protein [Arabidopsis thaliana] 125 318 10 PHE0004421_PMON951202.00E−53 78 33321848 gb|AAQ06658.1|apetala2 domain-containing CBF1- likeprotein [Oryza sativa] 126 319 10 PHE0004422_PMON95123 3.00E−51 8025991254 gb|AAN76804.1|DREB-like protein [Zea mays] 127 320 8PHE0004425_PMON94428 7.00E−37 98 11762134 gb|AAG40345.1|AT5g17460[Arabidopsis thaliana] 128 321 8 PHE0004431_PMON94398 1.00E−159 99557818 ref|NP_012214.1|Pho85p cyclin of the Pho80p subfamily, forms afunctional kinase complex with Pho85p which phosphorylates Mmr1p and isregulated by Pho81p; involved in glycogen metabolism, expression iscell-cycle regulated; Pcl7p [Saccharomyces cerevisiae] 129 322 8PHE0004432_PMON94112 0 100 15156338 ref|NP_354295.1| hypotheticalprotein AGR_C_2368 [Agrobacterium tumefaciens str. C58] 130 323 8PHE0004472_PMON94115 1.00E−128 100 16323494 ref|NP_187978.1|seven inabsentia (SINA) family protein [Arabidopsis thaliana] 131 324 14PHE0004472_PMON94126 1.00E−128 100 16323494 ref|NP_187978.1|seven inabsentia (SINA) family protein [Arabidopsis thaliana] 132 325 14PHE0004488_PMON95609 1.00E−123 100 21554344 ref|NP_198627.1|ASF1- likeanti-silencing family protein [Arabidopsis thaliana] 133 326 14PHE0004491_PMON95628 3.00E−12 45 14916641 dbj|BAB19648.1|preprophytosulfokine [Oryza sativa] 134 327 14 PHE0004492_PMON95614 0100 22331730 ref|NP_190653.2|phototropic- responsive NPH3 family protein[Arabidopsis thaliana] 135 328 10 PHE0004545_PMON95117 1.00E−106 10028973235 ref|NP_173200.1| ribosomal protein L14 family protein[Arabidopsis thaliana] 136 329 8 PHE0004574_PMON94433 0 100 16329404ref|NP_440132.1|transaldolase [Synechocystis sp. PCC 6803] 137 330 14PHE0004606_PMON95627 0 100 130709 pir||S29317 phosphoprotein phosphatase(EC 3.1.3.16) 1 - maize gb|AAA33545.1| protein phosphatase-1 138 331 8PHE0004620_PMON94189 1.00E−101 57 56421275 ref|YP_148593.1|6-phosphofructokinase (phosphofructokinase) (phosphohexokinase)[Geobacillus kaustophilus HTA426] 139 332 14 PHE0004620_PMON944421.00E−101 57 56421275 ref|YP_148593.1|6- phosphofructokinase(phosphofructokinase) (phosphohexokinase) [Geobacillus kaustophilusHTA426] 140 333 14 PHE0004622_PMON95621 0 100 10177836ref|NP_974942.1|F-box family protein [Arabidopsis thaliana] 141 334 8PHE0004626_PMON95101 0 88 50942161 ref|XP_480608.1|putativegamma-aminobutyrate transaminase subunit precursor isozyme 3 [Oryzasativa (japonica cultivar- group)] 142 335 8 PHE0004630_PMON94367 0 1007270516 emb|CAB80281.1|NAD+ dependent isocitrate dehydrogenase-likeprotein [Arabidopsis thaliana] 143 336 3 PHE0004634_PMON94385 1.00E−102100 61656127 ref|NP_176491.1|AP2 domain-containing transcription factor,putative [Arabidopsis thaliana] 144 337 2 PHE0004640_PMON95066 0 7334913436 ref|NP_918065.1|putative fatty acid condensing enzyme CUT1[Oryza sativa (japonica cultivar- group)] 145 338 8 PHE0004645_PMON946551.00E−136 100 18411867 ref|NP_565174.1|14-3-3 protein GF14 pi (GRF13)[Arabidopsis thaliana] 146 339 14 PHE0004645_PMON94685 1.00E−136 10018411867 ref|NP_565174.1|14-3-3 protein GF14 pi (GRF13) [Arabidopsisthaliana] 147 340 8 PHE0004647_PMON94651 1.00E−117 100 21554066pir||T02447 hypothetical protein At2g46000 Arabidopsis thaliana 148 34114 PHE0004647_PMON94688 1.00E−117 100 21554066 gb|AAM63147.1|unknown[Arabidopsis thaliana] 149 342 14 PHE0004650_PMON94686 1.00E−112 10067633514 gb|AAY78681.1|putative E3 ubiquitin ligase SCF complex subunitSKP1/ASK1 [Arabidopsis thaliana] 150 343 8 PHE0004652_PMON946571.00E−138 100 38603872 dbj|BAD43212.1|putativeglutamate/aspartate-binding peptide [Arabidopsis thaliana] 151 344 14PHE0004652_PMON94687 1.00E−138 100 38603872 dbj|BAD43212.1|putativeglutamate/aspartate-binding peptide [Arabidopsis thaliana] 152 345 8PHE0004687_PMON94669 7.00E−61 91 21592528 ref|NP_568396.1|ring-boxprotein-related [Arabidopsis thaliana] 153 346 10 PHE0004689_PMON95131 0100 7268004 emb|CAB78344.1|serine/threonine- specific protein kinase MHK[Arabidopsis thaliana] 154 347 10 PHE0004691_PMON95129 0 100 51978966emb|CAB61629.1| spermidine synthase 1 [Oryza sativa] 155 348 14PHE0004719_PMON94698 1.00E−147 100 28416631 ref|NP_564556.1|zinc finger(C3HC4-type RING finger) family protein [Arabidopsis thaliana] 156 349 8PHE0004719_PMON95089 1.00E−147 100 28416631 ref|NP_564556.1|zinc finger(C3HC4-type RING finger) family protein [Arabidopsis thaliana] 157 350 8PHE0004734_PMON94667 1.00E−87 100 5080771 ref|NP_172848.1| eukaryotictranslation initiation factor 5A-1/eIF- 5A 1 [Arabidopsis thaliana] 158351 10 PHE0004735_PMON95116 9.00E−88 100 21592652 ref|NP_177100.1|eukaryotic translation initiation factor 5A, putative/ eIF-5A, putative[Arabidopsis thaliana] 159 352 8 PHE0004739_PMON95110 1.00E−109 1006562282 emb|CAB62652.1|rac-like GTP binding protein Arac11 [Arabidopsisthaliana] 160 353 8 PHE0004753_PMON95105 0 100 6684442 ref|NP_178062.1|succinate-semialdehyde dehydrogenase (SSADH1) [Arabidopsis thaliana] 161354 8 PHE0004759_PMON95109 0 100 29824301 ref|NP_849582.1|expressedprotein [Arabidopsis thaliana] 162 355 10 PHE0004770_PMON95122 1.00E−3292 51038072 gb|AAT93875.1|unknown protein [Oryza sativa (japonicacultivar-group)] 163 356 10 PHE0004772_PMON95132 6.00E−36 33 9758946ref|NP_200265.1| expressed protein [Arabidopsis thaliana] 164 357 10PHE0004774_PMON95147 6.00E−52 66 50909195 ref|XP_466086.1|putativemultiple stress-responsive zinc-finger protein [Oryza sativa (japonicacultivar- group)] 165 358 10 PHE0004777_PMON95118 2.00E−64 100 26452894ref|NP_180514.1|DNA- directed RNA polymerase I(A) and III(C) 14 kDasubunit (RPAC14) [Arabidopsis thaliana] 166 359 14 PHE0004785_PMON950571.00E−145 84 34484312 sp|Q6UNT2|RL5_CUCSA 60S ribosomal protein L5 167360 10 PHE0004786_PMON95604 0 100 7267537 ref|NP_192634.1|phosphate-responsive protein, putative (EXO) [Arabidopsis thaliana] 168361 8 PHE0004788_PMON95092 0 84 31126776 ref|XP_506910.1| PREDICTEDOSJNBa0057G07.4 gene product [Oryza sativa (japonica cultivar-group)]169 362 10 PHE0004799_PMON95602 0 99 9843858 emb|CAC03739.1|flavincontaining polyamine oxidase [Zea mays] 170 363 10 PHE0004841_PMON956360 100 50909767 ref|XP_466372.1|cryptochrome 1a [Oryza sativa (japonicacultivar-group)] 171 364 10 PHE0004844_PMON95637 3.00E−53 100 62734659gb|AAX96768.1|expressed protein [Oryza sativa (japonica cultivar-group)]172 365 14 PHE0004854_PMON95611 1.00E−163 100 21592743ref|NP_199265.1|ribose 5- phosphate isomerase-related [Arabidopsisthaliana] 173 366 10 PHE0004862_PMON95601 5.00E−56 100 34902924dbj|BAB07982.1|FPF1 protein-like [Oryza sativa (japonicacultivar-group)] 174 367 10 PHE0004888_PMON95603 0 100 32405610ref|XP_323418.1|hypothetical protein [Neurospora crassa] 175 368 n/aAt1g21790.1 1.00E−168 100 21593249 ref|NP_564152.1|expressed protein[Arabidopsis thaliana] 176 369 n/a ERD4 0 100 17104683ref|NP_564354.1|early- responsive to dehydration stress protein (ERD4)[Arabidopsis thaliana] 177 370 n/a At1g78070.2 0 100 42572153ref|NP_974167.1|WD-40 repeat family protein [Arabidopsis thaliana] 178371 n/a At1g78070.1 1.00E−128 100 18411805 ref|NP_565168.1|WD-40 repeatfamily protein [Arabidopsis thaliana] 179 372 n/a At3g47340.1 0 1005541701 ref|NP_190318.1| asparagine synthetase 1[glutamine-hydrolyzing]/ glutamine-dependent asparagine synthetase 1(ASN1) [Arabidopsis thaliana] 180 373 n/a At3g47340.3 0 100 30692853ref|NP_850664.1|asparagine synthetase 1 [glutamine-hydrolyzing]/glutamine- dependent asparagine synthetase 1 (ASN1)[Arabidopsis thaliana] 181 374 n/a At3g47340.2 0 100 30692849ref|NP_850663.1|asparagine synthetase 1 [glutamine-hydrolyzing]/glutamine- dependent asparagine synthetase 1 (ASN1)[Arabidopsis thaliana] 182 375 n/a At5g13170.1 1.00E−163 100 9955561ref|NP_196821.1|nodulin MtN3 family protein [Arabidopsis thaliana] 183376 n/a At2g19900.1 0 100 28059162 ref|NP_179580.1|malateoxidoreductase, putative [Arabidopsis thaliana] 184 377 n/a At5g09480.18.00E−80 100 9955535 ref|NP_196510.1| hydroxyproline-rich glycoproteinfamily protein [Arabidopsis thaliana] 185 378 n/a At5g09530.1 0 1007671436 ref|NP_196515.1| hydroxyproline-rich glycoprotein family protein[Arabidopsis thaliana] 186 379 n/a At2g42790.1 0 100 21700853ref|NP_181807.1|citrate synthase, glyoxysomal, putative [Arabidopsisthaliana] 187 380 n/a At3g56200.1 0 100 7572918 ref|NP_191179.1|aminoacid transporter family protein [Arabidopsis thaliana] 188 381 n/aAt5g01520.1 1.00E−141 100 7327811 ref|NP_195772.1|zinc finger(C3HC4-type RING finger) family protein [Arabidopsis thaliana] 189 382n/a At5g01520.2 2.00E−97 100 7327811 ref|NP_195772.1|zinc finger(C3HC4-type RING finger) family protein [Arabidopsis thaliana] 190 383n/a At5g66780.1 2.00E−66 100 9758128 d ref|NP_201479.1| expressedprotein [Arabidopsis thaliana] 191 384 n/a At5g59320.1 1.00E−61 10024417292 ref|NP_568905.1|lipid transfer protein 3 (LTP3) [Arabidopsisthaliana] 192 385 n/a AtHB7 1.00E−151 100 20259175gb|AAM14303.1|putative homeodomain transcription factor protein ATHB-7[Arabidopsis thaliana] 193 386 n/a RD20 1.00E−136 100 20465881ref|NP_180896.1|calcium- binding RD20 protein (RD20) [Arabidopsisthaliana]

Screening Methods for Transgenic Plants with Enhanced Agronomic Trait

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Screening is necessary to identify the transgenic plant of thisinvention. Transgenic plants having enhanced agronomic traits areidentified from populations of plants transformed as described herein byevaluating the trait in a variety of assays to detect an enhancedagronomic trait. These assays also may take many forms, including butnot limited to, analyses to detect changes in the chemical composition,biomass, physiological properties, morphology of the plant. Changes inchemical compositions such as nutritional composition of grain can bedetected by analysis of the seed composition and content of protein,free amino acids, oil, free fatty acids, starch or tocopherols. Changesin biomass characteristics can be made on greenhouse or field grownplants and can include plant height, stem diameter, root and shoot dryweights; and, for corn plants, ear length and diameter. Changes inphysiological properties can be identified by evaluating responses tostress conditions, e.g., assays using imposed stress conditions such aswater deficit, nitrogen deficiency, cold growing conditions, pathogen orinsect attack or light deficiency, or increased plant density. Changesin morphology can be measured by visual observation of tendency of atransformed plant with an enhanced agronomic trait to also appear to bea normal plant as compared to changes toward bushy, taller, thicker,narrower leaves, striped leaves, knotted trait, chlorosis, albino,anthocyanin production, or altered tassels, ears or roots. Otherscreening properties include days to pollen shed, days to silking, leafextension rate, chlorophyll content, leaf temperature, stand, seedlingvigor, internode length, plant height, leaf number, leaf area,tittering, brace roots, stay green, stalk lodging, root lodging, planthealth, barreness/prolificacy, green snap, and pest resistance. Inaddition, phenotypic characteristics of harvested grain may beevaluated, including number of kernels per row on the ear, number ofrows of kernels on the ear, kernel abortion, kernel weight, kernel size,kernel density and physical grain quality.

Although preferred seeds for transgenic plants with enhanced agronomictraits of this invention are corn and soybean plants, other seeds arefor cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet,rice, tobacco, fruit and vegetable crops, and turfgrass

Example 1 Plant Expression Constructs

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic plants of this invention.

Primers for PCR amplification of protein coding nucleotides ofrecombinant DNA are designed at or near the start and stop codons of thecoding sequence, in order to eliminate most of the 5′ and 3′untranslated regions. Each recombinant DNA coding for a proteinidentified in Table 1 is amplified by PCR prior to insertion into theinsertion site of one of the base vectors as referenced in Table 5.

A. Corn Transformation Constructs

With reference to Table 2 and FIG. 1, pMON82060 illustrates the elementsof base vector 1 for corn transformation. Other base vectors for corntransformation were also constructed by replacing the gene of interestplant expression cassette elements of base vector 1, i.e. the promoter,leader, intron and terminator elements, with the elements listed inTable 5 to provide base vectors 2-12 for corn transformation. Each ofthe protein encoding DNA as identified in Table 1 is placed in the geneof interest plant expression cassette before the termination sequence ineach of the base vector 1-12.

TABLE 2 pMON82060 Coordinates of SEQ ID function name annotation NO:12603 Agro B-AGRtu.right Agro right border sequence, essential for5235-5591 transformation border transfer of T-DNA. Gene of P-Os.Ac1Promoter from the rice actin gene act1. 5609-7009 interest plantL-Os.Act1 Leader (first exon) from the rice actin 1 expression gene.cassette I-Os.Act1 First intron and flanking UTR exon sequences from therice actin 1 gene T-St.Pis4 The 3′ non-translated region of the7084-8026 potato proteinase inhibitor II gene which functions to directpolyadenylation of the mRNA Plant P-CaMV.35S CaMV 35S promoter 8075-8398selectable L-CaMV.35S 5′ UTR from the 35S RNA of CaMV markerCR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226expression resistance to neomycin and kanamycin cassette T-AGRtu.nos A3′ non-translated region of the 9255-9507 nopaline synthase gene ofAgrobacterium tumefaciens Ti plasmid which functions to directpolyadenylation of the mRNA . . . Agro B-AGRtu.left Agro left bordersequence, essential for  39-480 transformation border transfer of T-DNA.Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication from567-963 in E. coli plasmid RK2. CR-Ec.rop Coding region for repressor ofprimer 2472-2663 from the ColE1 plasmid. Expression of this gene productinterferes with primer binding at the origin of replication, keepingplasmid copy number low. OR-Ec.ori-ColE1 The minimal origin ofreplication from 3091-3679 The E. coli plasmid ColE1. P-Ec.aadA-SPC/promoter for Tn7 adenylyltransferase 4210-4251 STR (AAD(3″)) CR-Ec.aadA-Coding region for Tn7 4252-5040 SPC/STR adenylyltransferase (AAD(3″))conferring spectinomycin and streptomycin resistance. T-Ec.aadA- 3′ UTRfrom the Tn7 adenylyltransferase 5041-5098 SPC/STR (AAD(3″)) gene of E.coli.

Elements of a corn transformation plasmid, pMON17730, for expressing aLeuconostoc mesenteroides sucrose phosphorylase are illustrated in Table3. This construct was assembled using the technology known in the art.

TABLE 3 pMON17730 Coordinates of function name annotation SEQ ID NO:12606 Agro B-AGRtu.right Agro right border sequence, essential 4862-5218transformation border for transfer of T-DNA. Gene of P-Zm.Brittle2Promoter from thecorn brittle 2 gene 5276-6375 interest plant L-ZmBrittle2 5′ untranslated region from the corn 6385-7857 expressionbrittel 2 gene. 7870-8079 cassette L-Ta.Lhcb1 wheat CAB leader I-Os.Act1First intron and flanking UTR exon sequences from the rice actin 1 geneCR-Lm.spl1 PHE0004028_PMON17730 SPL coding region T-Ta.Hsp17 The 3′non-translated region of the wheat low molecular weight heat shockprotein gene Plant P-CaMV.35S CaMV 35S promoter 8226-8518 selectableCR-Ec.nptII- nptII selectable marker that confers 8583-9377 marker Tn5resistance to neomycin and expression kanamycin cassette T-AGRtu.nos A3′ non-translated region of the 9409-9661 nopaline synthase gene ofAgrobacterium tumefaciens Ti plasmid which functions to directpolyadenylation of the mRNA . . . Agro B-AGRtu.left Agro left bordersequence, essential 10003-10026 transformation border for transfer ofT-DNA. Maintenance OR-Ec.oriV- The vegetative origin of replication194-590 in E. coli RK2 from plasmid RK2. CR-Ec.rop Coding region forrepressor of 2099-2290 primer from the ColE1 plasmid. Expression of thisgene product interferes with primer binding at the origin ofreplication, keeping plasmid copy number low. OR-Ec.ori- The minimalorigin of replication 2718-3306 ColE1 from the E. coli plasmid ColE1.P-Ec.aadA- promoter for Tn7 3837-3878 SPC/STR adenylyltransferase(AAD(3″)) CR-Ec.aadA- Coding region for Tn7 3879-4667 SPC/STRadenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycinresistance. T-Ec.aadA- 3′ UTR from the Tn7 4668-4725 SPC/STRadenylyltransferase (AAD(3″)) gene of E. coli.

B. Soybean Transformation Constructs

Plasmids for use in transformation of soybean are also prepared.Elements of an exemplary common expression vector plasmid pMON82053 areshown in Table 4 and FIG. 2. Other base vectors for soybeantransformation were also constructed by replacing the gene of interestplant expression cassette elements of base vector 13, i.e. the promoter,leader, intron and terminator elements, with the elements listed inTable 5 to provide base vectors 13-15 for soybean transformation. Eachof the protein encoding DNA as identified in Table 1 is placed in thegene of interest plant expression cassette before the terminationsequence in each of the base vector 13-15.

TABLE 4 pMON82053 Coordinates of SEQ ID function name annotation NO:12604 Agro B-AGRtu.left Agro left border 6144-6585 transforamtion bordersequence, essential for transfer of T-DNA. Plant P-At.Act7 Promoter fromthe selectable arabidopsis actin 7 gene marker L-At.Act7 5′UTR ofArabidopsis 6624-7861 expression Act7 gene cassette I-At.Act7 Intronfrom the Arabidopsis actin7 gene TS-At.ShkG-CTP2 Transit peptide regionof 7864-8091 Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding8092-9459 CP4.nno_At region with dicot preferred codon usage. Gene ofT-AGRtu.nos A 3′ non-translated region 9466-9718 interest of thenopaline synthase expression gene of Agrobacterium cassette tumefaciensTi plasmid which functions to direct polyadenylation of the mRNA.P-CaMV.35S-enh Promoter for 35S RNA  1-613 from CaMV containing aduplication of the −90 to −350 region. T-Gb.E6-3b 3′ untranslated region 688-1002 from the fiber protein E6 gene of sea-island cotton; AgroB-AGRtu.right Agro right border 1033-1389 transformation bordersequence, essential for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2The vegetative origin of 5661-6057 in E. coli replication from plasmidRK2. CR-Ec.rop Coding region for 3961-4152 repressor of primer from theColE1 plasmid. Expression of this gene product interferes with primerbinding at the origin of replication, keeping plasmid copy number low.OR-Ec.ori-ColE1 The minimal origin of replication from the E. 2945-3533coli plasmid ColEl. P-Ec.aadA- romoter for Tn7 2373-2414 SPC/STRadenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn71584-2372 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycinand streptomycin resistance. T-Ec.aadA- 3′ UTR from the Tn7 1526-1583SPC/STR adenylyltransferase (AAD(3″)) gene of E. coli.

TABLE 5 Compositions of expression cassettes for gene of interest inplant transformation base vectors SEQ SEQ SEQ SEQ ID ID ID ID promoterNO leader NO intron NO terminator NO Base vector for corn 1 P-Os.Act112581 L-Os.Act1 12592 I-Os.Act1 12596 T-St.Pis4 12598 2 P-Hv.Per1 12582L-Hv.Per1 12593 I-Zm.DnaK 12597 T-St.Pis4 12598 3 P-Zm.RAB17 12591 NONE/ I-Zm.DnaK 12597 T-St.Pis4 12598 4 P-Zm.NAS2 12584 L-Zm.NAS2 12595I-Zm.DnaK 12597 T-St.Pis4 12598 5 P-Zm.PPDK 12585 L-Zm.PPDK 12588I-Zm.DnaK 12597 T-St.Pis4 12598 6 P-Os.GT1 12586 NONE / I-Zm.DnaK 12597T-St.Pis4 12598 7 P-Zm.PPDK 12587 L-Zm.PPDK 12588 I-Zm.DnaK 12597T-St.Pis4 12600 8 P-Os.Act1 12581 L-Os.Act1 12592 I-Os.Act1 12597T-St.Pis4 12598 9 P-Zm.PPDK 12587 L-Zm.PPDK 12588 I-Zm.DnaK 12597T-St.Pis4 12600 10  P-Os.Act1 12581 L-Os.Act1 12592 I-Os.Act1 12596T-St.Pis4 12598 11  P-Zm.SzeinC1 12589 L- 12601 I-Zm.DnaK 12597T-St.Pis4 12598 Zm.SzeinC1 12  P-Zm.NAS2 12584 L-Zm.NAS2 12595 I-Zm.DnaK12597 T-St.Pis4 12598 Base vector for Soybean 13  P-CaMV.35S- 12590 NONE/ NONE / T-Gb.E6 12599 enh 14  P-CaMV.35S- 12590 NONE / NONE / T-Gb.E612599 enh 15  P-Gm.Sphas 1 12583 L- 12594 NONE / T-Gb.E6 12599 Gm.Sphas1DNA constructs with some recombinant DNA of interest, e.g., SEQ ID NO:72, also contain a chloroplast transit peptide adjacent to therecombinant DNA.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton are also prepared. Elementsof an exemplary common expression vector plasmid pMON99053 are shown inTable 6 below and FIG. 3. Primers for PCR amplification of proteincoding nucleotides of recombinant DNA are designed at or near the startand stop codons of the coding sequence, in order to eliminate most ofthe 5′ and 3′ untranslated regions. Each recombinant DNA coding for aprotein identified in Table 1 is amplified by PCR prior to insertioninto the insertion site within the gene of interest expression cassetteof pMON99053

TABLE 6 Coordinates of function name annotation SEQ ID NO: 12606 AgroB-AGRtu.right Agro right border sequence, 11364-11720 transforamtionborder essential for transfer of T-DNA. Gene of interest Exp-CaMV.35S-Enhanced version of the 35S RNA 7794-8497 expression enh + ph.DnaKpromoter from CaMV plus the cassette petunia hsp70 5′ untranslatedregion T-Ps.RbcS2-E9 The 3′ non-translated region of the  67-699 peaRbcS2 gene which functions to direct polyadenylation of the mRNA. Plantselectable Exp-CaMV.35S Promoter from the rice actin 1  730-1053 markergene expression CR-Ec.nptII-Tn5 first exon of the rice actin 1 gene1087-1881 cassette T-AGRtu.nos A 3′ non-translated region of the1913-2165 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmidwhich functions to direct polyadenylation of the mRNA. Agro B-AGRtu.leftAgro left border sequence, 2211-2652 transformation border essential fortransfer of T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative originof 2739-3135 E. coli replication from plasmid RK2. CR-Ec.rop Codingregion for repressor of 4644-4835 primer from the ColE1 plasmid.Expression of this gene product interferes with primer binding at theorigin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1The minimal origin of replication 5263-5851 from the E. coli plasmidColEl. P-Ec.aadA-SPC/ romoter for Tn7 6382-6423 STR adenylyltransferase(AAD(3″)) CR-Ec.aadA-SPC/ Coding region for Tn7 6424-7212 STRadenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycinresistance. T-Ec.aadA-SPC/ 3′ UTR from the Tn7 7213-7270 STRadenylyltransferase (AAD(3″)) gene of E. coli.

Example 2 Corn Plant Transformation

This example illustrates the production and identification of transgeniccorn cells in seed of transgenic corn plants having an enhancedagronomic trait, i.e. enhanced nitrogen use efficiency, increased yield,enhanced water use efficiency, enhanced tolerance to cold and/orimproved seed compositions as compared to control plants. Transgeniccorn cells are prepared with recombinant DNA expressing each of theprotein encoding DNAs listed in Table 1 by Agrobacterium-mediatedtransformation using the corn transformation vectors 1-12 prepared asdisclosed in Example 1. Corn transformation is effected using methodsdisclosed in U.S. Patent Application Publication 2004/0344075 A1 wherecorn embryos are inoculated and co-cultured with the Agrobacteriumtumefaciens strain ABI and the corn transformation vector. To regeneratetransgenic corn plants the transgenic callus resulting fromtransformation is placed on media to initiate shoot development inplantlets which are transferred to potting soil for initial growth in agrowth chamber followed by a mist bench before transplanting to potswhere plants are grown to maturity. The plants are self fertilized andseed is harvested for screening as seed, seedlings or progeny R2 plantsor hybrids, e.g., for yield trials in the screens indicated above.

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants do not exhibit an enhanced agronomictrait. The transgenic plants and seeds having the transgenic cells ofthis invention which have recombinant DNA imparting the enhancedagronomic traits are identified by screening for nitrogen useefficiency, yield, water use efficiency, cold tolerance and improvedseed composition.

Example 3 Soybean Plant Transformation

This example illustrates the production and identification of transgenicsoybean cells in seed of transgenic soybean plants having an enhancedagronomic trait, i.e. enhanced nitrogen use efficiency, increased yield,enhanced water use efficiency, enhanced tolerance to cold and/orimproved seed compositions as compared to control plants. Transgenicsoybean cells are prepared with recombinant DNA expressing each of theprotein encoding DNAs listed in Table 1 by Agrobacterium-mediatedtransformation using the soybean transformation vectors 13-15 preparedas disclosed in Example 1. Soybean transformation is effected usingmethods disclosed in U.S. Pat. No. 6,384,301 where soybean meristemexplants are wounded then inoculated and co-cultured with the soybeantransformation vector, then transferred to selection media for 6-8 weeksto allow selection and growth of transgenic shoots.

The transformation is repeated for each of the protein encoding DNAsidentified in Table 1 in one of the base vectors 13-15.

Transgenic shoots producing roots are transferred to the greenhouse andpotted in soil. Many transgenic events which survive to fertiletransgenic plants that produce seeds and progeny plants do not exhibitan enhanced agronomic trait. The transgenic plants and seeds having thetransgenic cells of this invention which have recombinant DNA impartingthe enhanced agronomic traits are identified by screening for nitrogenuse efficiency, yield, water use efficiency, cold tolerance and improvedseed composition.

Example 4 Cotton Transgenic Plants with Enhanced Agronomic Traits

Cotton transformation is performed as generally described in WO0036911and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing therecombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 193are obtained by transforming with the cotton transformation vectoridentified in Example 1.

Progeny transgenic plants are selected from a population of transgeniccotton events under specified growing conditions and are compared withcontrol cotton plants. Control cotton plants are substantially the samecotton genotype but without the recombinant DNA, for example, either aparental cotton plant of the same genotype that was not transformed withthe identical recombinant DNA or a negative isoline of the transformedplant. Additionally, a commercial cotton cultivar adapted to thegeographical region and cultivation conditions, i.e. cotton varietyST474, cotton variety FM 958, and cotton variety Siokra L-23, are usedto compare the relative performance of the transgenic cotton plantscontaining the recombinant DNA. The specified culture conditions aregrowing a first set of transgenic and control plants under “wet”conditions, i.e. irrigated in the range of 85 to 100 percent ofevapotranspiration to provide leaf water potential of −14 to −18 bars,and growing a second set of transgenic and control plants under “dry”conditions, i.e. irrigated in the range of 40 to 60 percent ofevapotranspiration to provide a leaf water potential of −21 to −25 bars.Pest control, such as weed and insect control is applied equally to bothwet and dry treatments as needed. Data gathered during the trialincludes weather records throughout the growing season includingdetailed records of rainfall; soil characterization information; anyherbicide or insecticide applications; any gross agronomic differencesobserved such as leaf morphology, branching habit, leaf color, time toflowering, and fruiting pattern; plant height at various points duringthe trial; stand density; node and fruit number including node abovewhite flower and node above crack boll measurements; and visual wiltscoring. Cotton boll samples are taken and analyzed for lint fractionand fiber quality. The cotton is harvested at the normal harvesttimeframe for the trial area. Enhanced water use efficiency is indicatedby increased yield, improved relative water content, enhanced leaf waterpotential, increased biomass, enhanced leaf extension rates, andimproved fiber parameters.

Cotton plants with the transgenic cells by this invention are identifiedfrom among the transgenic cotton plants by agronomic trait screening ashaving increased yield and enhanced water use efficiency.

Example 5 Homolog Identification

This example illustrates the identification of homologs of proteinsencoded by the DNA identified in Table 1 which is used to providetransgenic seed and plants having enhanced agronomic traits. From thesequence of the homologs, homologous DNA sequence can be identified forpreparing additional transgenic seeds and plants of this invention withenhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequencesusing a proprietary sequence database and the National Center forBiotechnology Information (NCBI) non-redundant amino acid database(nr.aa). For each organism from which a polynucleotide sequence providedherein was obtained, an “Organism Protein Database” was constructed ofknown protein sequences of the organism; it is a subset of the AllProtein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences providedherein as SEQ ID NO: 194 through SEQ ID NO: 386 using NCBI “blastp”program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, andseparated by organism names. For each organism other than that of thequery sequence, a list was kept for hits from the query organism itselfwith a more significant E-value than the best hit of the organism. Thelist contains likely duplicated genes of the polynucleotides providedherein, and is referred to as the Core List. Another list was kept forall the hits from each organism, sorted by E-value, and referred to asthe Hit List.

The Organism Protein Database was queried using polypeptide sequencesprovided herein as SEQ ID NO: 194 through SEQ ID NO: 386 using NCBI“blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits werekept. A BLAST searchable database was constructed based on these hits,and is referred to as “SubDB”. SubDB was queried with each sequence inthe Hit List using NCBI “blastp” program with E-value cutoff of 1e-8.The hit with the best E-value was compared with the Core List from thecorresponding organism. The hit is deemed a likely ortholog if itbelongs to the Core List, otherwise it is deemed not a likely orthologand there is no further search of sequences in the Hit List for the sameorganism. Homologs from a large number of distinct organisms wereidentified and are reported by amino acid sequences of SEQ ID NO: 387through SEQ ID NO: 12580. These relationships of proteins of SEQ ID NO:194 through 386 and homologs of SEQ ID NO: 387 through 12580 isidentified in Table 7. The source organism for each homolog is found inthe Sequence Listing.

TABLE 7 SEQ ID NO: homolog SEQ ID NOs 196: 3549 1976 8970 12287 11799758 6083 9821 8256 7610 7869 4091 1111 1113 8630 7054 10917 3094 67129080 2702 2718 1130 1131 5382 6582 559 2169 1134 1132 1139 2295 116158090 2133 5063 5000 10336 12279 3828 7214 1485 2156 2232 2229 2242 22092203 2177 2207 2160 2151 11166 3220 197: 3549 1976 4850 8970 12287 11799758 6083 9821 4946 11935 8256 7610 7869 1841 9456 4091 1113 1111 86307054 7880 6876 6237 6712 9080 2702 2718 1130 1131 5382 6582 559 21691134 1132 1139 2295 11615 8090 5063 5000 10336 12279 3828 7214 1485 21562232 2229 2242 2209 2203 2207 2177 2160 2151 7622 1377 6970 6143 198:3549 1976 2210 6154 1028 1769 758 12325 9821 2973 4946 11935 8256 76105387 5384 5361 10434 8983 5051 4091 2766 6248 1113 1111 8630 9080 27022718 1131 1130 5382 7052 6582 1134 1132 1139 11615 2295 8090 6572 48031970 8113 3883 9565 1707 517 12372 11514 5441 5421 3828 7214 1485 1097199: 3549 4850 2210 8970 12287 2360 11500 11799 6912 1028 6154 758 57836083 9552 12325 9821 2973 4946 8256 7610 5387 5361 5384 5300 10434 89835051 1111 1113 8630 9080 2702 2718 1130 1131 5382 7052 6582 559 21691134 1132 1139 8090 6572 11350 7138 1730 10762 11345 527 8679 5063 50002879 517 7986 12372 11514 10336 6955 12279 5441 5421 3828 7214 1485 22292207 2242 2232 2209 2203 2156 2160 2151 5328 8248 200: 11500 5617 81503321 2181 4364 1769 1028 5122 11328 6042 2711 1760 4874 4098 1914 118537334 6504 10624 2638 11705 7913 12171 12198 10430 12189 12219 1040410432 10408 6957 8282 6184 11935 580 10470 1940 11039 8629 1096 74212505 5801 11671 4006 12473 6778 2607 10849 6279 7500 2657 5584 80592622 2043 3269 10363 6186 9631 9243 11098 1168 6690 8584 10577 687 29779804 9337 6306 9118 4356 10225 9740 6652 5251 12514 7463 706 3048 37801925 11765 9803 10824 3004 5275 8642 1664 12173 4049 2031 11681 89802339 9172 11955 10576 9333 10482 813 5656 4628 10843 2352 5484 2856 43132877 1633 11143 6066 7722 7746 10941 11741 2941 2745 11364 7638 78841328 5606 6580 11262 7483 8156 412 453 7288 6842 1286 7896 9734 657010595 8863 1246 7112 12464 1373 3779 2705 5044 4017 5712 4619 3539 10291610 5976 4964 11724 9037 8989 1126 4073 395 10344 5428 4845 1611 104844496 3517 3418 10294 2427 3442 9747 5534 9571 1125 9720 9319 12346 34171588 2779 4611 5312 10179 6867 3049 3051 9900 1265 9463 4576 764 6024432 8921 11379 2141 1755 9498 7395 8179 7462 7279 8729 9676 11351 175810907 4995 1205 608 12100 8331 8341 10326 6852 11947 6597 2475 6407 807710788 11815 5269 489 9317 5574 11240 11821 11485 2868 9753 676 112231924 8045 1689 12035 11980 5906 7805 6728 5177 1711 1715 5050 1601 112421010 11286 7814 7152 3730 5888 615 11078 9681 2883 8522 8210 4450 116327573 6031 2713 3861 9480 5307 7874 2048 5136 8625 2168 4580 10634 57725082 8731 2678 9311 10561 7803 4408 6227 12026 11234 7247 5578 9683 39992953 2193 3370 11542 10711 6403 4207 11251 8447 6805 727 951 737 90901828 1928 2277 986 739 7044 10025 7409 9449 944 8427 10911 3965 12995294 6332 5145 9418 6150 9008 1004 3831 5157 6968 11922 7392 9855 50615448 6857 2354 2879 620 7986 10208 4520 9003 8015 525 8013 11884 1072612493 9260 8508 5693 1450 4258 201: 7470 10842 5790 6772 1530 9966 997310368 655 4677 4157 1015 9967 9732 1621 1702 2553 11599 9342 3724 66134462 2681 4577 3827 8039 2557 8538 9605 12321 3228 2139 9255 11428 30225404 9564 12166 8047 11255 11888 1492 5870 4250 554 12481 8585 5674 20622021 6718 2810 4015 12306 8941 3135 7850 7009 4247 5760 643 2512 24228709 5661 2437 11487 8706 3703 6811 5006 6000 2290 11973 8426 2912 649810642 8257 5362 1189 996 1740 10904 5778 4372 12095 1616 9708 1598 45257513 1934 10939 9044 7273 6105 6950 12122 5936 2802 3711 8640 6644 98426994 2587 7510 8609 1877 5408 8009 9943 8475 4333 8476 2651 5379 11144202: 7477 1676 4448 2400 6045 6940 8526 9923 11995 8913 10513 634 796911746 6446 4371 1018 4026 10874 11604 5505 9219 4140 11205 12025 36051669 1987 2822 2279 10124 11930 4546 3504 1950 7696 1604 4492 710 117373171 8574 11646 9030 765 203: 9581 1789 9205 10127 507 7859 5085 107942201 5072 1384 7541 12225 5253 4000 8561 1469 3834 12504 9837 7137 46709143 1972 230 11807 7457 3867 12503 9644 10286 686 3416 8708 913 93919343 1949 971 11938 12315 7511 9076 8346 3455 1790 6685 11054 10989 47759544 2197 3225 1198 7996 9715 6751 11217 3189 10361 3589 2768 4753 3937426 9423 2744 1339 10139 2332 8771 3079 4312 7098 11256 1681 642 4115179 11964 5793 8376 2386 9500 2401 5669 10501 1939 11311 4977 7401 826612472 480 10947 12116 539 7591 1020 1493 9017 2513 3100 4405 5679 33733795 10805 11445 10653 5898 5556 12139 12448 8448 5245 12533 10039 13242498 9955 6104 5516 204: 3474 7088 4085 10331 6972 7065 2023 10909 59155913 6491 5970 6936 5920 5919 5966 5944 5738 5968 6663 1233 5947 225810694 9592 4692 12344 11227 6753 8618 205: 9144 6127 6445 4401 3645 97565274 8302 1548 9875 9979 1922 1941 9100 7274 12121 11051 11528 9523 78303543 6760 1979 3997 9779 9635 4955 1818 946 5201 12580 8270 10531 4154910 1802 2256 2979 7899 3139 3777 10332 10536 4842 8280 9000 1327 109508576 513 4263 6884 8684 3877 7243 7262 6420 1424 2680 10546 9965 117116656 1164 1160 5248 4812 11605 3598 8386 12446 3922 10305 467 5963 94811998 4655 4064 446 6112 6111 4689 3743 449 1123 11231 11434 2456 47111629 6249 2152 2171 6494 8636 11953 5487 7844 6164 11566 1495 4623 69203447 3181 3153 1081 11890 3476 1127 1195 1192 12349 3600 11090 5377 80227160 11091 10643 7586 12247 6202 6217 4617 2237 2380 6219 1756 7456 9253237 206: 5804 12016 10678 10712 10735 7448 9024 10738 10708 12014 106387423 7421 7417 309 10586 10603 10589 10584 7444 10644 7446 5047 1064510646 12280 207 9710 2096 11839 9709 1612 8993 10037 6780 11613 9034 306307 2004 11103 8166 6931 7311 6922 8933 10494 3783 308 11857 12034 3781916 6666 9745 9140 6285 12449 10356 9452 4275 12246 9728 9405 2987 72232067 3934 8138 11430 9052 12318 6252 410 2407 6792 3564 2073 4786 113269877 3397 310 11058 9105 8474 12047 6860 7715 860 8446 4050 6973 67259408 4088 3842 1902 4332 2342 1701 10402 11870 4672 3986 10725 121811973 3950 9992 4578 10224 862 7045 11785 4789 5465 8088 3553 10189 99642793 6677 10001 3375 4200 10391 1361 1234 10741 10641 10683 11712 1074310575 10581 4747 207: 12016 7448 12014 7423 309 5047 9710 2096 118391612 8993 10037 6780 9034 307 306 2004 6931 6922 8933 10494 308 118573781 916 9745 6666 9140 6285 12449 9452 8035 10356 11492 12021 444310064 8344 2067 3934 4275 8138 12246 9405 2987 9728 11430 9052 123186252 7223 410 6792 3564 2073 4786 9877 3397 310 11058 8474 9105 1187012047 4672 3986 6860 7715 860 2342 8446 1701 4050 10402 6973 9408 40883842 4332 6725 1902 10725 12181 6779 1973 2823 9849 10154 862 7045 117854789 5465 10224 8088 4578 10189 3553 2793 6677 10001 3375 4200 103913950 9992 1361 1468 9964 7410 2176 10741 11712 10743 10575 10581 206208: 8564 10720 7580 12251 9922 5975 8617 4257 645 3210 4615 8228 7471408 10412 3357 4397 7547 10137 3018 7289 11413 1687 2058 4738 127412252 8769 6626 4708 275 11442 2843 10230 6198 10814 2304 9207 209: 93868213 8184 6094 8240 8242 8209 8211 5327 9254 10652 9428 11965 1181211814 9275 9274 6208 8173 7971 9276 9278 9280 9297 9253 8100 9330 93039305 4986 4730 10770 11755 3994 5070 7569 5734 3989 3985 9531 9214 94299365 11108 6372 5373 2117 3351 12521 4075 1896 3535 10982 4340 2371 8583813 10602 5493 5548 10627 5552 2460 4278 1787 3297 2964 2965 2962 36309434 3625 4592 10087 8272 3870 4415 8484 5940 10629 10623 10636 1017410667 5553 10670 10671 2562 2568 8456 5226 5200 11493 7169 9374 796211722 5462 2866 10150 10170 10153 4425 1856 4727 9772 6514 2550 93674482 9458 9455 2869 2162 9300 9302 10632 3616 210: 2857 3612 6601 11831181 1182 6604 1159 10118 10806 11819 11745 6639 11715 7049 10888 100247122 8076 8876 8903 1266 10535 624 7532 4011 5266 6168 6326 11178 26412461 6646 8758 7990 9318 8505 7393 2727 6008 3940 9115 5137 9096 11481363 10193 9377 9250 5445 11200 11273 11276 211: 11176 8570 11245 102746081 7181 6450 4624 9320 6129 984 7196 7388 2804 542 11805 212: 23933407 11789 8124 2340 3714 1395 1433 12303 375 2814 6364 9438 3292 123902984 6746 9695 675 2101 3618 12081 6128 1892 3448 9864 6152 2844 73814291 4973 5447 10140 11877 8566 7624 6472 10665 2089 9925 938 8536 615610608 11433 5967 1511 11974 12573 4734 11501 5076 12428 8275 2769 440211854 213: 4784 5997 2399 6338 3933 4092 10151 2740 10610 214: 108552954 6766 2958 8910 12101 6783 3620 7658 7785 3180 9266 9246 9247 17928649 5777 10173 10178 3461 9046 5810 5806 1226 3287 12557 8375 122358403 8384 7414 5429 4396 6501 8433 7094 8413 7920 5588 9853 6890 64839273 9841 683 9313 6871 6899 6877 2491 4890 9129 5744 9572 11085 1203711048 12113 613 9424 6574 12066 7504 5863 8409 4273 10572 5923 1895 18939040 3665 5481 7755 8408 924 1454 12140 8378 5510 5509 5513 3124 310311911 4141 2082 2247 4630 8299 6667 702 8975 6801 745 741 779 770 772744 771 11549 719 7117 5565 11875 215: 11919 9154 5594 10308 2827 28303408 3403 2471 5367 1120 5371 5081 4880 10931 7367 6883 11808 6136 254911638 6868 8315 3118 10508 10877 650 5616 4115 3026 3028 9516 785 90837596 8108 4176 6525 5765 3802 1806 8081 7208 8893 12007 8654 9048 90728575 8423 6300 6409 4165 6095 9477 2485 10112 5117 2278 2281 2264 22846055 2348 4251 8187 10826 9660 9216 2777 4403 7239 2643 782 2262 81111799 781 2696 8265 821 6575 9029 6259 5907 2153 9132 1008 9697 116585996 6135 3512 216: 1063 9995 9748 8083 4921 10081 2976 7153 8380 10722845 2124 5604 2742 217: 1063 9995 9748 8083 4921 10081 2976 7153 83801072 2845 2124 5604 2742 218: 10265 3604 11692 2087 2100 2084 4972 86274940 10555 4941 652 1430 11778 7581 915 1478 8934 1244 9538 6106 95406923 5854 6892 9462 3486 10996 12018 9346 3284 6742 8247 219: 5171 345110952 6452 5333 11383 12420 9816 9099 11249 528 11871 11060 8935 35213063 10253 9510 10954 6303 6941 523 904 5364 4534 1993 9623 3245 125068843 10612 7200 2319 7201 1746 9164 1043 220: 6376 1316 5391 12526 71942996 3154 10569 11756 11824 3924 9004 5150 5993 10023 5309 10233 55829183 5649 2780 11917 6719 11145 10056 2516 1372 5622 7269 2665 1402 58857636 6193 3223 2719 6657 1867 7660 12334 9360 5492 2710 2076 8465 757111887 2033 8847 3260 10323 11018 7553 6905 5747 10773 5018 9023 94209484 9512 8291 2650 4553 2233 4983 7834 11916 8565 4123 1090 3981 6102885 5427 3349 649 9974 10523 10337 5840 8815 6996 11041 1321 1153211331 9757 6755 2327 2730 5199 5280 11943 3656 6297 4570 2983 6557 121452376 7618 6924 9049 10975 8678 12452 7263 2204 3741 7210 7502 4325 114081350 6089 2892 8054 8643 2501 1647 11693 6378 1729 6966 8734 9027 88275647 9075 7286 659 4113 6496 4454 11650 4378 2224 2687 1763 830 32555001 3830 6495 3121 1757 7740 8530 2770 1866 459 2049 4814 12517 24088583 6850 7550 5545 2042 3709 5474 11062 4761 10345 7778 1449 1562 89014943 10916 11403 6820 3167 1997 7484 9833 12022 8573 5100 5639 7158 87919723 4484 10282 1334 11312 11317 4294 9400 4982 7125 2655 10854 9131 9925153 2528 12519 12187 6818 799 861 11120 11361 6634 12230 10852 88173105 9513 8235 221: 6205 11358 3072 2888 2907 6203 2800 7221 4750 362712485 2816 10896 4463 3774 8273 5002 4122 8581 8364 3273 6044 6503 64516887 4226 5120 9987 679 12019 1695 939 9726 8964 2326 6178 6080 855112220 926 10271 3458 983 6773 5354 551 12326 1673 10474 7111 503 32617427 7498 5710 9522 12089 8842 8147 8799 9369 2355 6063 3582 3537 35571618 2519 10121 9781 10031 1438 4529 11657 7069 3979 1260 8752 9515 176210093 875 12460 12052 9166 7493 12523 10742 10451 8622 8931 10210 76683177 3657 6276 625 6423 222: 9766 2574 8653 12518 6881 10011 1281 44353555 696 5489 10478 6961 6001 1591 1453 10635 2267 6727 12366 4551 18891367 1388 9264 8099 5016 1033 4094 12546 7145 6511 1331 1524 3894 19438569 11313 5235 223: 12210 2632 5689 5995 9108 6848 3162 5357 9825 60999769 11406 12011 4089 11037 2154 7634 2930 6937 224: 11851 9599 392 35145363 9918 7949 12550 981 8255 3499 2997 9043 6076 2056 2922 11064 111319209 5316 10222 11118 1947 4743 225: 12336 2351 3767 1826 226: 917412242 516 9436 5692 6101 8462 9960 3910 227: 882 7014 8781 8246 107052703 8520 6497 7900 6599 3575 3216 228: 2359 5356 6318 6123 588 79086312 4748 9929 6824 6509 229: 9744 8168 1420 6853 7687 2503 7653 5252787 6057 2759 8114 9054 8122 8127 4410 5238 4675 7892 11484 12365 117443437 6705 3241 11187 230: 9581 9205 10794 5085 2201 1384 4000 8605 46701972 10286 11576 686 913 2768 203 393 4753 1339 2332 8771 9423 7426 27443079 10139 4312 11256 7098 642 1681 411 11964 5179 1324 9955 6104 55169644 11807 7457 231: 12356 4958 6943 8532 8516 9081 4754 8450 8451 106774939 12575 11787 7205 4213 972 3291 9604 11517 7192 10860 5598 125384035 11116 695 7007 479 4154 10733 232: 641 10835 7416 7705 8597 55065365 2998 4911 1710 4507 7519 1965 233: 7211 12486 7508 11321 5086 118188707 9321 1682 4612 3885 10374 3698 2956 2709 2789 9060 3654 4690 90897726 3369 8385 2927 2192 5052 11202 11758 10190 5874 8038 8631 537 106554768 2120 3687 4281 11320 6521 4769 7545 7786 7407 12108 9206 12454 21477282 12432 3610 8128 5956 3069 234: 9373 9421 11561 11557 12294 103019284 6616 1308 6809 3915 11093 3919 11088 11597 11298 11592 6281 391711137 1726 11130 1230 3689 4740 3725 11047 2975 6172 1216 3544 4142 7375746 11962 6474 12427 235: 9373 9421 11561 11557 12294 10301 9284 66161308 6809 3915 11093 3919 11088 11597 11298 11592 6281 3917 11137 172611130 1230 3689 4740 3725 11047 2975 6172 1216 3544 4142 7375 746 119626474 12427 236: 444 10758 1559 12502 889 9874 9788 7310 12020 6831 798010109 5949 6731 11689 7825 3697 1264 4393 548 2268 1773 3208 1147 40299056 1141 7469 5188 10443 7314 1452 1744 5383 237: 5650 5881 10697 33432506 6706 9195 3119 609 11113 12263 12264 9501 8410 8925 3221 7983 7956933 2361 8269 9921 6336 10563 632 12541 10155 10751 9511 7976 6351 548210797 4571 1776 12112 7190 1900 9324 6339 7001 2317 9820 7015 6384 491711822 4227 11377 6229 10949 11498 1448 8172 10908 7776 6183 238: 365111823 2950 1915 5176 4381 8742 6316 9780 3427 8319 899 4829 11372 122326415 3788 1658 9838 11020 8918 7485 10102 8428 1054 2552 11363 124899487 10566 9535 11344 4210 1739 5067 8368 9789 7897 2937 10388 885910675 3146 1783 2989 3471 4847 919 918 5832 1172 2121 5023 806 1145912478 12285 11359 2683 11412 12180 11214 5716 7022 8289 6594 7858 112701848 12273 9776 6464 1578 4239 7235 5329 9074 3608 6048 1812 3310 78725540 8662 4796 790 2336 6532 8866 6741 7383 5683 4201 1638 1583 681911937 2788 11593 12298 6125 6977 1956 8141 7002 1569 11618 3937 564810925 10480 9137 6221 2366 6277 10503 5161 12302 5628 4791 239: 365111823 2950 1915 5176 4381 8742 6316 9780 3427 8319 899 4829 11372 122326415 3788 1658 9838 11020 8918 7485 10102 8428 1054 2552 11363 124899487 10566 9535 11344 4210 1739 5067 8368 9789 7897 2937 10388 885910675 3146 1783 2989 3471 4847 919 918 5832 1172 2121 5023 806 1145912478 12285 11359 2683 11412 12180 11214 5716 7022 8289 6594 7858 112701848 12273 9776 6464 1578 4239 7235 5329 9074 3608 6048 1812 3310 78725540 8662 4796 790 2336 6532 8866 6741 7383 5683 4201 1638 1583 681911937 2788 11593 12298 6125 6977 1956 8141 7002 1569 11618 3937 564810925 10480 9137 6221 2366 6277 10503 5161 12302 5628 4791 240: 52983673 6171 5229 8230 6271 9427 1356 10882 11852 10687 6088 10076 983010597 6373 3987 10322 241: 2393 3407 11789 11391 11346 5568 689 91213768 6558 5447 1870 7849 2504 8733 10066 994 11743 980 4909 7933 84868369 5152 1705 6156 10608 242: 3431 5395 4346 8330 8327 8702 7787 52658943 12561 4536 11625 4411 1035 11796 6078 2720 4449 10010 3057 98763536 5603 11727 5025 698 9899 6457 10804 3454 2741 11343 11668 125379198 9194 6906 11749 2886 4118 11050 3125 3104 8238 7647 11157 115525735 3190 1224 2010 10669 3186 12278 10534 9546 10088 3888 1521 1062610413 11620 12324 1406 12498 3067 7386 6359 10120 6004 2803 9290 11141854 2391 6032 10433 12371 11636 11795 6713 8567 10754 717 2465 9545 98866990 4012 8324 3742 1053 8586 8683 10073 12149 7481 2755 2646 6082 89564440 4579 4447 6886 4268 561 11512 3439 1568 8328 1091 7948 5861 726 58211893 2118 12271 6845 6843 6847 2068 9119 2022 8587 12175 8754 6777 4979325 6872 7531 11335 2928 9885 5358 1963 6109 7533 11337 11444 9889 61791632 6874 3342 12072 12199 11476 9892 6224 6254 5355 11338 7625 1042610428 11395 1315 11066 11063 11076 5351 1440 11336 11316 3538 8749 97785154 9356 835 831 2589 8503 10727 2891 8958 11046 6790 9818 9094 98285374 8610 7368 244: 9283 4282 1754 571 9388 10252 4060 4063 10254 939210256 10251 8049 10250 10237 10240 8942 10188 9163 9457 9412 4148 27952440 10370 3577 434 12383 7841 4935 4928 4931 4933 4908 701 7640 26028966 4824 5455 4822 4823 5454 9020 8692 9551 6293 8301 3456 1197 124213247 6475 7319 9389 569 6169 1497 1499 9460 2309 600 2172 2178 6997 26688309 2821 4458 2940 9832 5380 6918 732 9375 9376 9294 2109 12129 811712379 4965 10550 7218 12182 11653 9067 4197 9956 4905 3379 8543 566310834 8946 3814 4643 1788 6732 9857 4189 10740 6347 1240 5417 6399 17811782 3068 3398 10951 3402 4557 3406 1307 4161 11960 8337 8307 7812 24862510 1431 6430 894 5282 12422 6029 10938 10935 10936 2671 4260 108917914 5646 9237 11207 11111 4869 10685 6067 4596 12455 4572 7777 20721364 3840 4879 1193 7010 1667 2748 4809 1850 8067 8922 9994 5724 121606849 3432 11503 11999 2619 11453 5074 12050 3193 3298 5344 3303 33013300 3362 3283 3251 3211 9607 3277 4512 10924 8703 8838 4726 6467 2527962 5203 4589 9809 9806 9805 9812 12423 12314 3674 3728 3676 4145 76904132 3000 3002 2009 2415 3348 2543 1825 3368 1753 11838 6270 11163 473611353 3911 10235 10232 9385 10283 10275 10280 10279 1285 1293 1279 13121291 1089 2708 1407 10065 9873 12041 9138 11097 4104 405 10732 384310734 9232 1527 5586 4841 245: 9283 4282 1754 571 9388 10252 4060 406310254 9392 10256 10251 8049 10250 10237 10240 8942 10188 9163 9457 94124148 2795 2440 10370 3577 434 12383 7841 4935 4928 4931 4933 4908 7017640 2602 8966 4824 5455 4822 4823 5454 9020 8692 9551 6293 8301 34561197 12421 3247 6475 7319 9389 569 6169 1497 1499 9460 2309 600 21722178 6997 2668 8309 2821 4458 2940 9832 5380 6918 732 9375 9376 92942109 12129 8117 12379 4965 10550 7218 12182 11653 9067 4197 9956 49053379 8543 5663 10834 8946 3814 4643 1788 6732 9857 4189 10740 6347 12405417 6399 1781 1782 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4287 7838 8011 5651 5941 9051 5564 4621 7863 2536 5059 57735837 12335 6680 7099 7118 10998 2432 2463 1750 9628 6813 3628 9033 583610449 5420 5207 3387 7412 3524 394 11949 8547 4036 6873 1600 1443 53494233 9783 7318 2864 11057 1323 3052 10007 2599 6329 9359 7436 7657 118791025 12197 9682 10707 9968 4445 801 8656 6142 2948 7184 4255 10302 1036412386 7309 12216 6438 2050 3050 2920 5965 11072 9831 3854 5662 120033011 2728 9738 1017 8134 526 363: 6176 8639 1152 1522 8352 6375 102015139 8072 2308 4150 3445 7428 2024 4633 4866 2581 12337 12476 10035 815311036 3036 7174 5414 3759 9595 1795 7561 5236 11547 1813 1163 4779 36954760 6556 8661 11299 11986 8914 10050 11070 4068 809 4568 3229 102634659 10993 8382 1047 2931 9895 4478 1570 7237 4269 3838 1016 4137 1118312276 11482 9854 11264 3433 7046 8216 3513 9383 8148 1602 6058 2215 18425999 10212 8258 6591 4686 9520 8497 6648 3088 6786 5986 4715 7335 65101839 5658 2103 11352 7577 4634 7828 9426 11791 11970 560 12269 104543529 7253 5891 880 4009 2760 7540 9015 8638 3356 5775 10574 1338 121173249 7644 9762 3751 7042 3672 4202 5589 6019 9835 7229 7693 3086 102969608 11707 2588 6425 9954 7071 10204 7525 6469 3192 5805 6230 4385 21806991 11841 9662 5353 2773 10791 3059 6304 7745 5886 6518 1536 1863 13855022 6003 8904 7257 9755 11607 11221 6807 4696 8329 9798 1221 364: 47146552 6036 9637 11177 7157 2425 8841 4455 8468 3444 8404 3936 763 109421668 885 1786 10802 7271 8055 4969 11623 3054 11828 2575 5685 5924 54604819 9989 11834 10380 5520 9589 3334 4912 9894 7974 8700 12342 365: 45439647 3507 1211 3246 10549 2019 2094 4177 4338 9238 5770 4772 6343 404812231 10905 12008 4926 12442 2270 5780 11506 7565 1094 12147 1451 4733294 11314 9173 2035 2593 12094 5494 5488 3087 2026 8206 9878 4705 104724746 6642 5937 8790 11896 2244 5026 5835 7953 11936 10110 7133 3785 366:10341 5696 6298 2325 2938 8027 1580 7429 589 12057 10353 3976 3860 16469225 842 5818 4573 1901 8803 367: 4208 3058 2394 8507 2917 6904 134510919 4336 1202 7187 1114 3353 3660 2387 8437 9496 9413 11082 8079 120402841 4934 12221 8040 4875 7674 11844 10774 6033 11664 5283 5192 10853967 2564 3007 11334 11393 9940 872 2508 12169 3721 6130 1306 5401 10879883 9814 5802 10180 6895 7491 3952 4386 8159 1106 5271 6278 4124 116126546 556 9556 5366 11798 6295 10902 3531 3572 3574 7418 3765 2343 73566349 9808 3624 9583 6431 3128 8098 3871 2494 1319 2271 4787 1838 14565064 6878 9972 8908 6774 3809 8730 11094 670 1496 5212 3588 9459 85186963 10018 7791 8264 2418 6442 11211 1636 6014 5278 4155 11100 1052811481 10313 7126 8887 2600 11160 3425 5846 4898 4527 6393 12267 174710239 10330 3670 10329 10327 11203 10715 8883 10249 1046 11155 5228 20702128 7076 6412 6875 5853 8795 564 9262 2640 5083 1875 6898 6951 123737127 368: 6754 10585 6689 9826 964 4693 8822 6462 691 2410 9712 98843219 7232 12539 1991 11008 9322 8725 369: 1461 7305 10596 7966 8323 90364856 433 2663 8335 8509 11775 10231 5820 8056 12556 5630 11866 9292 2220509 12153 7303 12393 7926 370: 1200 12300 576 5306 8372 2320 7732 67883633 10013 657 1403 10074 7663 6585 8051 2569 2722 8325 1121 11616 42486146 4276 2647 5766 9625 10767 6273 4186 6188 3988 4877 3998 8151 60182040 595 371: 1200 12300 6283 5306 1573 8372 9432 1142 533 2320 7732 3701267 595 3852 372: 7477 1676 4448 2400 6045 6940 9923 11995 8913 10513634 7969 11746 6446 4371 1018 4026 10874 11604 5505 9219 4140 6427 120251669 3605 5027 10847 11128 1483 9362 2044 12343 3560 430 11220 1214610479 7769 4704 5385 12424 3286 1435 12461 10990 7985 5652 6086 120672785 6367 3470 2598 10591 4756 2959 10900 11878 12536 5795 12559 54508391 10046 7073 3157 2834 8144 2236 2782 1926 9617 1987 2822 2279 219610312 5369 10124 11903 12289 4170 12092 8540 2362 12010 5204 9431 66203500 9585 7989 1508 4885 877 6119 3807 2585 12330 4380 3820 5302 30566337 2060 3096 202 4492 1604 11737 710 8574 11646 3171 9030 765 568010955 6592 2624 9114 5576 5196 5912 1980 11275 1119 3929 9810 1209911930 4546 3504 1950 7696 374 1703 373: 7477 1676 4448 2400 6045 69409923 8913 10513 634 11746 6446 4371 1018 4026 11604 5505 9219 4140 642712025 3605 11128 9362 1483 5027 10847 2044 12343 3560 430 11220 1214612424 3286 12461 10479 10990 5652 5385 6086 4704 7769 7985 2785 636710591 10900 11878 12536 12559 12067 10046 3157 5450 8144 9617 1987 282210312 2196 5369 10124 11903 12289 2362 12010 6620 5204 7989 4885 8774170 2585 8540 4380 12092 3820 1508 3056 6337 5302 3096 2060 202 44921604 11737 710 8574 11646 3171 9030 765 5680 2624 9114 6592 10955 51965912 1980 11275 9810 12099 4821 10994 8060 1531 6262 4632 1058 2716 73174825 3731 5786 414 5576 1000 9714 1854 2489 4638 4485 11930 3504 19507696 374 372 1703 374: 7477 1676 4448 2400 6045 6940 9923 11995 891310513 634 7969 11746 6446 4371 1018 4026 10874 11604 5505 9219 4140 642712025 3605 1669 10847 5027 11128 9362 1483 2044 12343 3560 430 1122012146 10479 4704 7769 5385 12424 3286 12461 10990 7985 5652 6086 143512067 2785 6367 3470 10591 2959 10900 2598 11878 12536 12559 5450 100467073 3157 2834 5795 8144 9617 10146 1987 2822 2279 2196 10312 5369 1012411903 12289 4170 2362 12010 5204 6620 12092 8540 9585 9431 3500 79891508 4885 877 2585 4380 3820 3056 6337 5302 2060 3096 202 4492 160411737 710 8574 11646 3171 9030 765 5680 6592 10955 2624 9114 5196 59121980 11275 5576 9810 12099 11930 4546 3504 1950 7696 372 375: 2393 340711789 2984 10140 7809 9834 6717 6156 10608 6364 376: 6589 11497 683611332 11382 5877 2213 2951 8215 1064 1057 8227 8249 10551 3319 8195 82188221 9142 9661 8198 6116 10889 10520 7399 3659 1734 5077 3510 5010 83519403 9244 483 4466 12167 7031 4216 6477 4107 12150 6828 5305 12404 15677248 11614 7445 3482 5079 2734 11033 3311 7402 4221 2223 1040 9289 29024438 4600 4324 9168 2576 10382 7057 4976 1719 10894 10483 3361 9016 56347496 1784 9760 12134 7188 8533 10833 1303 9336 4810 10290 3800 111926040 852 586 9907 11699 8192 7242 2963 10418 12069 9588 10604 2330 92426800 10782 7530 6261 8220 8962 7198 7306 7697 2102 2085 9372 5757 370011432 3755 11635 3376 11831 9573 10934 12553 9010 12441 2757 3093 119139725 3144 5892 10167 7130 5337 3872 1625 4143 7507 3179 7982 2590 12031932 1225 6260 1044 9032 498 499 9053 7023 8322 4927 3331 10393 813211728 8655 7771 988 8909 1791 5413 2157 10976 1365 2511 3200 7167 998611603 1041 6238 10166 11045 9704 9621 7669 2014 3281 11206 2697 40675118 12401 5368 12284 9430 7136 12412 6387 9543 2522 572 5119 1606 839410248 11146 10285 11719 6776 11309 11139 10439 9245 11197 11729 20364759 9773 6077 5571 11810 10084 10857 8191 9597 8037 3279 8225 797 976977 979 999 5185 4166 9598 4901 7135 2944 10328 8189 6781 377: 105276513 5978 2596 1034 5101 1637 6141 6160 5334 12110 378: 11873 5442 79397815 10258 11591 11595 4116 4129 9663 4114 11588 11586 11585 9690 115564111 11550 9687 4264 11554 5405 4130 3884 9667 3491 11545 3879 115463875 9646 3873 4105 3881 4101 4086 4084 3850 4082 4078 8588 8545 85899271 8300 5033 5035 5058 1832 5005 9380 5056 2097 1720 7704 10811 27335138 3490 8251 3805 2314 3573 5858 2753 2629 7295 557 3786 8599 91532012 5249 12190 6141 5334 6162 7529 1731 10108 463 12105 5048 660 773110191 9632 11189 6866 10706 4292 7977 437 10106 5124 1552 2131 765111225 8644 1923 3585 6432 3532 379: 8061 8064 7961 7964 404 3270 112153265 10192 11241 2078 11168 7871 12087 4499 1544 1412 6268 8820 11677914 5507 4996 4991 4416 4419 1981 4347 3401 10092 11767 11031 12403 97998861 3101 12558 10974 1155 1157 1162 1171 1167 10984 5609 5612 5156 58095813 2069 10182 4042 3226 7512 6791 5053 6674 11820 11674 10970 946611069 10506 2468 2442 2444 2291 2435 2288 2294 2296 1196 12392 1241412417 12418 12430 12435 12437 12453 8217 3323 4516 10500 12038 2221 58343035 7400 2413 1207 2833 2850 4552 4556 3578 5581 555 7183 552 7176 5537179 549 7185 4518 4522 4539 4541 3889 12474 5841 9042 5812 8199 82008494 469 4590 6355 711 9845 9847 4984 7919 7432 7433 4285 11121 45037100 5439 2561 5559 1944 1237 10062 2402 4735 6583 3891 5718 5725 57469737 2946 2949 7280 5538 2658 2674 9057 8712 9135 5144 6967 11626 107599277 11254 11258 10005 400 1615 4506 2137 6636 12186 843 8021 3464 433512045 3009 6698 7468 1733 3239 4185 12228 1011 4131 5279 11790 5453 54529366 10461 6840 5340 4790 9069 4421 767 6182 2807 11421 6683 6090 425411473 4256 7225 11457 6361 5623 1560 2751 6147 6148 6151 11409 1141111415 9097 1310 3111 4301 9508 7650 1796 11061 11104 11934 6530 906310105 4959 7479 8137 11271 4864 11951 8624 8110 5704 4119 6365 9492 796310912 2447 3204 3317 2521 2451 2450 2546 2514 3202 2487 2365 3205 23633148 2396 3354 2480 4047 3352 3203 3175 3243 2484 3262 3173 2419 23353380 3316 4051 3164 9189 11975 11977 12013 7351 6499 6517 11125 108809640 9651 5762 5769 11996 11736 6987 8226 3336 8224 1210 722 7083 114185972 9384 11424 6600 7623 7528 10940 10803 8430 3662 3099 4359 5542 168611972 8902 2017 2110 6669 6692 11541 4284 1473 4147 8879 8882 4376 85359364 10522 7701 8652 11989 10456 4561 12185 1501 3769 9669 5220 52189616 2405 12056 491 492 8338 819 2222 6325 1906 9158 6199 421 5392 24033750 694 697 699 724 728 6340 3693 3696 3955 7148 1166 2645 2409 87774990 8278 8279 8281 8304 8306 8308 8312 947 4453 1206 10609 10755 1173511539 11786 1546 10196 7632 3082 3037 3110 3106 6524 3289 12083 1208512082 490 6197 12080 12079 2219 8066 6405 8894 7333 6385 10897 8930 12233912 11910 1912 8891 3475 4698 2662 901 1194 1503 1507 8696 9203 6471566 6133 3248 11892 5459 3302 8553 4703 8621 851 4337 4341 3675 29705601 10607 9524 12425 7307 9223 3918 736 729 961 10464 6196 1543 27582971 5700 12211 2578 10438 3880 1539 8865 8981 3738 12457 445 3540 114565703 4762 8680 2839 583 4627 8732 5599 2623 6321 5659 10004 10168 87416218 2194 2214 6554 6244 6555 2191 7472 8949 2896 6699 6703 3469 49029719 1995 5537 3957 3964 11679 3719 424 8848 3586 11832 7055 10785 95077643 10467 7642 10395 8808 9495 8628 9050 12192 9591 3681 7246 1125710605 9721 2379 1187 1674 10009 1108 6522 3363 3360 1049 1051 1056 77567738 8897 2974 8924 2988 7438 832 4666 11859 4702 2991 4355 8867 49688868 3897 8895 2967 4669 4697 4674 4700 9415 6838 6549 2617 621 183110829 10808 4794 6627 4776 7605 8694 12200 11056 11002 7868 3699 2610419 418 10435 936 2345 6762 12543 9549 10661 10689 10699 10719 1190 11651161 1170 11929 11419 7988 3798 1218 1220 2721 1800 6167 1268 3178 380:7583 12223 3882 6192 10967 5287 11700 9299 12204 3071 9116 6233 36266939 5957 2854 2852 10080 6245 9582 11285 9334 2429 381: 611 12004 68145320 1380 5991 7802 5112 6551 402 7832 382 11138 382: 611 12004 59916551 7832 383: 4837 10247 4513 8974 9193 3250 11577 10663 2910 9363 1186384: 11191 6118 591 9506 9370 8717 1055 9504 10360 6606 575 5878 59113404 1764 7501 4369 4367 12240 12238 2227 7439 2003 7437 7440 7521 118622334 12466 12137 5673 11590 12090 11766 7515 5121 3935 6239 6232 8366307 6289 6328 6310 9157 8674 2257 2828 2851 6282 1990 10944 5126 5127905 3876 907 8578 912 8514 8899 12309 12059 10972 11683 4862 4099 119009518 9521 4985 9517 6702 960 9819 958 965 963 2731 11043 10487 1048611010 7682 1759 9618 9619 5901 10142 7649 753 755 4752 6402 11686 1413656 5951 9563 795 8523 4826 11233 3115 385: 12282 3816 6979 4487 81944174 1027 3606 9593 8612 10293 4967 3304 6074 6908 8506 6658 7332 86829519 4399 4231 3446 3597 11520 11676 11673 11704 11678 11714 11703 509010122 4379 386: 3874 2573 7549 7517 9650 3332 1953 4431 11909 3497 100452551 2545 9861 8988 4509 11527 10422 4863 8961 11568 5627 2724 9699 56758512 2715 4144

Example 6

This example illustrates the preparation and identification by screeningof transgenic seeds and plants having enhanced agronomic traits usingDNA encoding homologs identified in Example 7. Transgenic corn, soybeanor cotton seed and plants with recombinant DNA encoding each of thehomologs identified in Example 5 are prepared by transformation. Thetransgenic seed, plantlets and progeny plants are screened for nitrogenuse efficiency, yield, water use efficiency, growth under cold stressand seed composition change. Transgenic plants and seed having at leastone enhanced agronomic trait of this invention are identified.

Example 7

This example illustrates the identification of consensus amino acidsequence for the proteins and homologs encoded by DNA that is used toprepare the transgenic seed and plants of this invention having enhancedagronomic traits.

ClustalW program was selected for multiple sequence alignments of theamino acid sequence of SEQ ID NO: 371 and 11 homologs. Three majorfactors affecting the sequence alignments dramatically are (1) proteinweight matrices; (2) gap open penalty; (3) gap extension penalty.Protein weight matrices available for ClustalW program include Blosum,Pam and Gonnet series. Those parameters with gap open penalty and gapextension penalty were extensively tested. On the basis of the testresults, Blosum weight matrix, gap open penalty of 10 and gap extensionpenalty of 1 were chosen for multiple sequence alignment. Attached arethe sequences of SEQ ID NO: 371, its homologs and the consensussequence, SEQ ID NO: 12608 at the end. The symbols for consensussequence are (1) uppercase letters for 100% identity in all positions ofmultiple sequence alignment output; (2) lowercase letters for >=70%identity; symbol; (3) “X” indicated <70% identity; (4) dashes “-”meaning that gaps were in >=70% sequences.

SEQ ID NO:   371MDIFDNSDLEYLVDEFH--ADFDDDEPFGEVDVTSESDSDFMDSDFDFELSESKTNNETS 12300MDIFDNSDLEYLVDDFHGFSDSEDDEPFGEFDHKSEADSDFEDDLDPTQESD------TS  6283MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT  1573MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT  8372MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT  5306MEHFNNDDLEYVVDEYYDVPDFAVEDTS---SDIVPELTSDVDSDFEDEFPTSNAKTDTT  9432------------------------------------------------------------   533------------------------------------------------------------  2320------------------------------------------------------------  1142-------------------------------------------------MTISNTSSTSK  1200------------------------------------------------------------  7732-----------------MAHDLHDDLEFVSGDDDDYYLEFDHDPGHGFHTSAATSASQTL 12608xxxxxxxxxxxxxxxxxxxxxxxxxxxx---xxxxxxxxxxxxxxxxxxxxxxxxxxxxxALEARNGKDIQGIPWESLNYTRDRYRENRLLHYKNFESLFRSREELDKECLQVEKGKNFYALEARNGKDIQGIPWERLNYSRDQYRYKRLQQYKNFEILFRSRQDLDKECLQVEKGKHFYASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFYASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFYASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFYASEARNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECLEVQKGETFY-----------GIPWERLNYSRDKYRETRLKQYKNYQNFSRSRHDLRKECFEVQKGETFY-----------GIPWERLNYSRDKYRETRLKQYKNYQNFSLSPHHLHKECFQVQKGQTFY-----------GIPWERLNYSRDKYRETRLKQYKNYQNFSRSPHHLRKECFQVQKGQTFYTIFRRNGKDIQGIPWERLNYSRDKYRETRLKQYKNYQNFSLSPHHLHKECFQVQKGQTFY------------IPWERLQITRKDYRKARLEQYKNYENFPQSGELMDKLCKQVESSSKYYIGALYFRTSRWTIPWERLNYSRNQYREMRLRQYKNYENLTMPRDGLEKECKQVERKDTFYxxxxxxxxxxxgIPWErLnysRdxYRexRLxqYKNyxnfxxsxxxlxKeCxxVxkgxtfYDFQFNTRLVKSTIAHFQLR----------------NLVWATSKHDVYFMNNYSLMHWSSLDFQFNTRLVKSTIAHFQLR----------------NLLWATTKHDVYFMKNYSLMHWSSLDFFFNTRLVKSTIVHFQLR----------------NLLWATSKHDVYFMQNYSVMHWSALDFFFNTRLVKSTIVHFQLLRQVXVSSLAGPNIMLRNLLWATSKHDVYFMQNYSVMHWSALDFFFNTRLVRXTLAGPNIMLR--------------NLLWATSKHDVYFMQNYSVMHWSALDFFFNTRLVKSTIVHFQLRPN----------IMLRNLLWATSKHDVYFMQNYSVMHWSALDFFFNTRLVKSTIVHFQLR----------------NLLWATSKHDVYFMQNYSVMHWSALDFFFNTRLVKSTIVHFQLRN----------------LLWATSKHDVYLMQNYSVMHWSALDFFFNTRLVKSTIVHFQLQLGRTX-------IMLRNLLWATSKHDVYLMQNYSVMHWSALDFFFNTRLVKSTIVHFQLLXRWNMSSLAGPYIMLRNLLWATSKHDVYLMQDYSVMHWSALEFQYNTRIVKPSILHFQLR----------------NLLWATSKHDVYFMSNSTVGHWSSLDFHLNTRLVKSTIVHFQLR----------------NLLWATSKHDVYLMQNYSVMHWSSLdFxfNTRlVkstixhfqlxxx----------xxxxnLlWATsKHDVYxMqnysvmHWSxLLQRGKEVLNVAKPIVPSMKQHGSLSQSVSRVQISTMAVKDDLKLREGSKESLSVRKSTNLLQRSKEVLNVAKPIVPTMKQPGLLSQSISRVQISTMAVKDDLIVAGGFQGELICKRINEPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGX-SRVSLYNLKHPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHPLQRSKEVLNVAKPIIPTLTHPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKQPLRRGKEVLNVAKPIIPTLKRPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKNLKHPLQRSKEVLNVAKPIIPTLTHPGFLAQPVSRVQISTMTVKENLMVAGGFQGELICKVGLIISHKMTDVLDFSGHVAPAKKHPGCALEGFTGVQVSTLAVNEGLLVAGGFQGELVCKSLGERLQRGKEVLNVAGQLAPSQNVR--GAMPLSRVQISTMAVKGNLMVAGGFQGELICKYVDKPlxrxkeVLnvakpixPtxkxpgxlagpvsrVQiSTmxVkenLmvagGfqgelickxxxxpRLLSALN-----------------------------------------------------GVAFCTVLHRFX-NDITNSVDIYNAPSGSLRVITANNDCTVRVLDAXNFAFLNSFTL---GVLFCGKITTDDNAITHAV-DVYSNPAGSLRVITANNDFQGRVFD---------------GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGWFKYDWSGVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWSGVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWSGVLFCGKITTDDNAITNAV-DVYRNPAGSEGNPA--------------------------GVLFCGKITTDGNAITNAVXDVYRNPAGSLRVITAXNDSQASGFDAENFAS---------GVLFCGKITTDDNAITNAV-DVYSNPAGSLRVITANNDFQVRVFDAENFASLGCFKYDWSISYFHSI-----------------------------------------------------DVKFCTRTTLSDNAITNAM-DIHRSTSGSLRITVSNNDSGVREFDMERFQLLNHFRFNWPGVAFCTNLTGNNNSITNAV-DIYQAPNGGTRITTANNDCVVRTFDTERFSLISHFAFPWSgvxfcxxxtxxxnxitxax-dxyxxpxgsxrxxxxxndxxxxxxdxxxxxxxxxxxxxxx------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------VNNTSVSPDG--------------------------------------------------VNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQVNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQ------------------------------------------------------------------------------------------------------------------------VNNTSVSPDGKLLAVLGDSTECLIADANTGKITGSLKGHLDYSFSSAWHPDGQILATGNQ------------------------------------------------------------VNHTSVSPDKKLLAVVGDDRDALLVDSRNGKVTSTLVGHLDYSFASAWHLDGVTFATGNQVNNTSVSPDGKLLAVLGDSSDCLIADSQSGKEMARLKGHLDYSFSSAWHPDGRVVATGNQxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------DKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQDKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQ------------------------------------------------------------------------------------------------------------------------DKTCRLWDIRNLSQSMAVLKGRMGAIRALRFTSDGRFLAMAEPADFVHIFDSHSGYEQGQ------------------------------------------------------------DKTCRVWDIRNPSTSLAVLRGNIGAIRCIRYSSDGRFLLFSEPADFVHVYSTAECYRKRQDRTCRVWDVRNMSRSVAVLEGRIGAVRGLRYSPDGRFLAASEPADFVHVYDAAAGYADAQxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx------------------------------------------------------*------------------------------------------------------*------------------------------------------------------*------------------------------------------------------*EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------*EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------*------------------------------------------------------*------------------------------------------------------*EIDLFGEIAGISFSPDTEALFVGIADRTYGSLLEFNRKRHYNYLDSF-------*------------------------------------------------------*EIDFFGEISGISLSPDD------ESLFVGVCDRVYASLLNYRLVHANGYLDSYM*EIDLFGEIAGVAFSPAGNNGGGGEALFVSIADRTYGSLLEFHRRRRHGYLDCYV*xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx-------*

The consensus amino acid sequence can be used to identify DNAcorresponding to the full scope of this invention that is useful inproviding transgenic plants, e.g., corn, soybean and cotton plants withtransgenic cells expressing protein encoding DNA that impart an enhancedagronomic traits. For example, enhanced nitrogen use efficiency,enhanced yield, enhanced water use efficiency, enhanced growth undercold stress and/or improved seed compositions are imparted by theexpression in the plants of DNA encoding a protein with amino acidsequence identical to the consensus amino acid sequence.

Example 8 Identification of Target Genes of Transcription Factors ABF3and CBF3 Chemical Kinetics Models to Identify Regulator-TargetRelationships

It has been shown both in mRNA blotting and microarray experiments thatactivation of regulators under stress conditions usually occurs earlierthan that of its targets (Haake, 2002, Seki, 2002a). In eukaryoticcells, the effect of a regulator is usually achieved in multiple steps,including transcription of the regulator genes, transportation of theregulator mRNA(s) out of the nucleus, translation of the transcript(s),transportation of the regulator protein back to the nucleus, and thebinding of the regulator protein to the promoter regions of its targetgenes to achieve transcriptional regulation. Noticeable timingdifference exists among changes in concentrations of the regulator mRNA,the regulator protein, and the mRNAs of its targets. A chemical kineticsmodel naturally fits this context by taking into account of the timelags among these events.

Because the active level of the regulator protein is not measureddirectly in microarray experiments, the regulator protein concentrationis treated as a hidden variable in our model to serve as the linkbetween the measurable mRNA concentrations of a regulator and itstarget(s). More specifically, the regulator protein concentration can bemodeled by the following chemical kinetic equation without consideringpost-translational regulation:

$\begin{matrix}{\frac{R_{p}}{t} = {{K_{tran}R_{m}} - {K_{p}R_{p}}}} & {{equation}\mspace{14mu} (1)}\end{matrix}$

where R_(p) is the regulator protein concentration; R_(m) is theregulator mRNA concentration; K_(tran) is the apparent rate of mRNAtranslation, and K_(p) is the turnover rate of the regulator protein.Accordingly, the time course of the target mRNA concentration can bemodeled with the following equation

$\begin{matrix}{\frac{T_{m}}{t} = {B_{t} + {f\left( R_{p} \right)} - {K_{t\;}T_{m}}}} & {{equation}\mspace{14mu} (2)}\end{matrix}$

where T_(m) is the concentration of the target mRNA; B_(t) is the basaltranscription rate of the target gene; and K_(t) is the turnover rate ofthe target mRNA; f (R_(p)) measures the regulated transcription rate,which is different for activators and repressors. For activators, it hasthe following Taylor first order approximation when R_(p) is small (Chenet al., 1999).

$\begin{matrix}{{f\left( R_{p} \right)} = {{{f\left( {R_{p} = 0} \right)} + \frac{\left( {f\left( R_{p} \right)} \right.}{R_{p}}}_{{Rp} = 0}R_{p}}} & {{equation}\mspace{14mu} (3)}\end{matrix}$

f(R_(p)=0) is equal to zero, assuming target gene transcription shouldnot be activated when there is no regulator protein.

$\frac{\left( {f\left( R_{p} \right)} \right.}{R_{p}}_{{Rp} = 0}$

is the activation rate of regulator protein on the target gene. If it isreplaced by parameter K_(act) for simplicity, f (R_(p)) takes thefollowing form:

f(R _(p))=K _(act) R _(p)  equation (4)

The basal level target transcription rate should satisfy the followingcondition:

B _(t) +f(R _(pbasal))−K _(t) T _(m basal)=0  equation (5)

Where R_(pbasal) and T_(mbasal) are the basal concentrations of theregulator protein and target mRNA, respectively.

Usually, what is reported in transcription profiling experiment is notthe absolute concentration of mRNA, but rather a fold change compared tobasal transcription level of that gene. Thus, we define relative changesof R_(m) and T_(m) as R_(m)′ and T_(m)′

R _(m) ′=R _(m) /R _(mbasal)−1  equation (6)

T _(m) ′=T _(m) /T _(mbasal)−1  equation (7)

Combining equation (1), (2), (4), (5), (6) and (7), and considering thefact that K_(tran)R_(mbasel)−K_(p)R_(pbaseal)=0, leads to the followingsecond order ordinary differential equation:

$\begin{matrix}{{\frac{^{2}\left( T_{m}^{\prime} \right)}{t^{2}} + {\left( {K_{t} + K_{p}} \right)\frac{\left( T_{m}^{\prime} \right)}{t}} + {K_{t}K_{p}T_{m}^{\prime}}} = {\gamma \; R_{m}^{\prime}}} & {{equation}\mspace{14mu} (8)}\end{matrix}$

Where γ=K_(act)K_(tran)R_(mbasal)/T_(mbasel)

Given all the model parameters, the relationship between the relativemRNA levels of regulator and its target, R_(m)′ and T_(m)′, is definedby Equation (8). In other words, for the target gene of a regulator, itsrelative mRNA level T_(m)′ has to satisfy equation (8), given the modelparameters and the relative regulator mRNA level R_(m)′. It isinteresting to note that the regulator protein concentration, a keyvariable in the original model equations, is not involved explicitly inthe final equation relating the relative mRNA levels of regulator andtarget. To predict the target of a specific regulator, we can solveequation (8) to obtain the theoretical target behavior curve, and thenfind the genes with mRNA levels similar to the theoretical curve, whichwill be identified as the potential targets of that regulator.

In the case of transcript expression profiling experiments under stressconditions, the initial conditions should be the following:

$\begin{matrix}{{T_{m}^{\prime}_{t = 0}} = 0} & {{equation}\mspace{14mu} (9)} \\{{\frac{\left( T_{m}^{\prime} \right)}{t}_{t = 0}} = 0} & {{equation}\mspace{14mu} (10)}\end{matrix}$

Because the target gene mRNA and the regulator protein should be attheir basal levels at the onset of stress condition (t=0). It isapparent from equations (2) and (5) that initial condition (10) shouldbe true.

To approximate R_(m), a stepwise linear model can be fit as follows:

R _(m)′_(i)(t)=α_(i)+β_(i) tt _(i) ≦t≦t _(i+1) i=0, . . . ,n−1  equation (11)

Where t_(i) is i^(th) time point; and α_(i) and β_(i) are the parametersof stepwise linear function in each time interval, which are determinedby the measured regulator mRNA levels at the two adjacent time points.Equation (8) has analytic solution:

Tm _(i)(t)′=A _(i) e ^(−K) ^(t) ^(t) +B _(i) e ^(K) ^(p) ^(t) +D _(i) tt_(i) ≦t≦t _(i+1) ,i=0, . . . , n−1  equation (12)

Where D_(i)=β_(i)γ/K_(p)K_(t) andC_(i)=[α_(i)γ−(K_(p)+K_(t))D_(i)]/K_(p)K_(t)The contiguous restrictions on T_(m)′ are stated in the followingequations:

$\begin{matrix}{{{{Tm}_{i}^{\prime}(t)} = {{Tm}_{i + 1}^{\prime}(t)}},{{{when}\mspace{14mu} t} = {{t_{i}\mspace{14mu} i} = 1}},\ldots \mspace{14mu},{n - 1.}} & {{equation}\mspace{14mu} (13)} \\{{\frac{\left( {{Tm}_{i}^{\prime}(t)} \right)}{t} = \frac{\left( {{Tm}_{i + 1}^{\prime}(t)} \right)}{t}},{{{when}\mspace{14mu} t} = {{t_{i}\mspace{14mu} i} = 1}},\ldots \mspace{14mu},{n - 1.}} & {{equation}\mspace{14mu} (14)}\end{matrix}$

After substituting equation (12) into equations (9), (10), (13) and(14), A_(i) and B_(i) can be obtained by solving sets of linear algebraequations, and are functions of α_(i), β_(i), γ, K_(t) and K_(p).

Learning model parameters. For each regulator and target pair, there arethree parameters involved in equation (8), the target mRNA turnover rateK_(t), the active regulator turnover rate K_(p), and γ, which is equalto K_(act)K_(tran)R_(mbasal)/T_(mbasel). K_(act) represents the strengthof regulator protein effect on the target gene; K_(tran) is thetranslation rate of regulator mRNA. They lump together with the ratio ofbasal mRNA concentrations of regulator and target to form parameter γ,which determines the magnitude of the relative target mRNA level but notits shape. It is the parameters K_(t) and K_(p) that determine the shapeof the relative target mRNA level, such as how fast the target generesponds to the regulator.

For gene expression experiments under stress conditions in plants, thekinetics model can be trained with known regulator-target pair reportedin the literature (e.g., CBF and RD17 in Arabidopsis under cold stress)with a non-linear regression model. When the normalized expressionprofile of a target gene with its maximal response is considered, thereis no need to keep γ as a free model parameter (γ1=nγ2 leads toT_(m1)′=nT_(m2)′ when other parameters are kept the same in equations(8), (9) and (10)). Therefore, only two parameters K_(t) and K_(p) areestimated from the non-linear regression model, and are used to predictother regulators and their targets in plant stress response. Thetheoretical target mRNA expression profiles are calculated for all thegenes annotated as transcription factors, and Pearson correlationcoefficient is computed for each theoretical target profile and eachobserved expression profile in each stress condition. When highcorrelation in one or several stress conditions is found, thetranscription factor could be one of the putative regulators of thecorresponding gene.

Target gene prediction using promoter motif analysis. As an additionalline of evidence for regulator-target pair prediction, we used promotermotif analysis to correlate regulators and their potential targets.Differentially expressed genes under stress conditions measured inmicroarray experiments can be partitioned into certain number ofclusters based on the similarity in their expression profiles. All knownpromoter motifs within 1500 base-pairs distance to the starting codonwere extracted from AGRIS database (Davuluri, 2003) for each gene. Thefrequency of each promoter motif in each cluster is computed, andFisher's Exact Test is conducted to test the over-representation ofcertain promoter motifs. Enriched promoter motifs for a given clusterare selected as putative regulator motifs when statistical significancemeets certain cutoff value (e.g., p-value 0.05). When a transcriptionfactor (or a family of transcription factors) is known to bind to theputative regulator motif, the transcription factor(s) should be theputative regulators of target genes with the regulator motif in thatcluster.

Combining evidences from kinetics models and promoter analysis. Kineticsmodels and promoter analysis independently predict putativeregulator-target pairs, we attempted to combine their results to enhanceour ability to detect true regulator-target pairs. In our kineticsmodels, for each target gene only the transcription factors with aPearson correlation coefficient higher than certain cutoff in at leastone stress condition are considered as its potential regulators. It ispossible that the same regulator regulates its target genes in differentstress conditions. Therefore, it is reasonable to give a higher rankingfor a regulator if its theoretical target profiles are correlated tothose of certain gene in multiple conditions. Based on these ideas, aranking score for each possible regulator-target pair is derived asfollows:

$\begin{matrix}{{{score}\left( {r_{i},t_{j}} \right)} = {\prod\limits_{k}^{\;}\; {{R_{k}\left( {r_{i},t_{j}} \right)}/N}}} & {{equation}\mspace{14mu} (15)}\end{matrix}$

Where R_(k)(r_(i),t_(j)) is the rank of Pearson correlation coefficientof the theoretical target profile of transcription factor r_(i) to thatof gene t_(j) in stress condition k; N is the total number oftranscription factors on DNA chip.

The rank of the scores for putative transcription factors shouldrepresent the likelihood of them being the true regulator for a specificgene. Similarly, the rank of p-value of motif enrichment is theindicator of the likelihood of a transcription factor(s) being the trueregulator for a specific target. Lastly, we combine both rankings fromkinetics model prediction and promoter analysis by defining a score fora given regulator-target pair as following:

$\begin{matrix}{{{L\left( {r_{i},t_{j}} \right)} = {{\prod\limits_{m}^{\;}{{{{rank}_{m}\left( {r_{i},t_{j}} \right)}/N}\mspace{14mu} m}} = 1}},2} & {{equation}\mspace{14mu} (17)}\end{matrix}$

Where L(r_(i),t_(j)) can be viewed as the strength of transcriptionfactor r_(i) to be the regulator of gene t_(j); rank₁(r_(i),t_(j)) andrank₂(r_(i),t_(j)) are the rank of score(r_(i),t_(j)) from kineticsmodel prediction, and the rank of p-value of regulator r_(i) bindingmotifs enrichment for the cluster with gene t_(j), respectively.

This method was applied to an Arabidopsis gene expression datasetmeasuring responses to various stress conditions (Seki et al., 2002a;Seki, et al., 2002b). In this experiment, wild-type Arabidopsis plantswere subject to stress treatments for various periods (1, 2, 5, 10 and24 hours), and extracted mRNA samples were hybridized to a cDNAmicroarray with ˜7000 full-length cDNAs. 493 genes were chosen for theanalysis, as each of these genes was differentially regulated in atleast one of the stress conditions. Table 8 shows the evidence of thepredicted target genes of CBF3 in terms of evidence strength, whetherevidence from kinetics model or enriched promoter analysis exists foreach predicted target.

TABLE 8 Evidence SEQ ID strength Kinetics Promoter NO Target (10⁻⁵)model Analysis / At1g01470 2.13333 yes yes / AtGolS3 2.13333 yes yes /RD17 2.13333 yes yes / ERD10 2.13333 yes yes 175 At1g21790 2.13333 yesyes / ERD7 2.13333 yes yes / cor15A 2.13333 yes yes / FL3-5A3 4.26667yes yes / kin2 4.26667 yes yes / cor15B 6.4 yes yes 176 ERD4 26.66667yes no / RD29A 26.66667 yes no / At1g16850 53.33333 yes no 177 and 178At1g78070 800 no yes / kin1 800 no yesTable 9 shows the evidence of the predicted target genes of ABF3 interms of the evidence strength, whether evidence from kinetics model orenriched promoter analysis exists for each predicted target.

TABLE 9 Evidence strength Kinetics Promoter SEQ ID NO Target (10⁻⁵)model Analysis 179, 180 and 181 At3g47340 1.26222 yes yes 182 At5g131701.26222 yes yes 183 At2g19900 1.26222 yes yes 184 and 185 At5g095302.52444 yes yes 186 At2g42790 2.52444 yes yes 187 At3g56200 2.52444 yesyes 188 and 189 At5g01520 2.52444 yes yes 190 At5g66780 3.78667 yes yes191 At5g59320 3.78667 yes yes 192 AtHB7 5.04889 yes yes / RD29B 7.57333yes yes 193 RD20 7.57333 yes yes

It has been shown that ABF3 and CBF3 confer stress tolerance totransgenic plants. Thus, the target genes of ABF3 and CBF3, identifiedby this invention, including SEQ ID NO: 368 through SEQ ID NO: 386, andtheir homologs, are particularly useful for producing transgenic plantcells in crop plants with enhanced stress tolerance.

Example 9 Identification of Amino Acid Domain by Pfam Analysis

The amino acid sequence of the expressed proteins that were shown to beassociated with an enhanced trait were analyzed for Pfam protein familyagainst the current Pfam collection of multiple sequence alignments andhidden Markov models using the HMMER software in the appended computerlisting. The Pfam protein families for the proteins of SEQ ID NO: 194through 386 are shown in Table 10. The Hidden Markov model databases forthe identified patent families are also in the appended computer listingallowing identification of other homologous proteins and their cognateencoding DNA to enable the full breadth of the invention for a person ofordinary skill in the art. Certain proteins are identified by a singlePfam domain and others by multiple Pfam domains. For instance, theprotein with amino acids of SEQ ID NO: 194 is characterized by threePfam domains, i.e. PPDK_N, PEP-utilizer and PEP-utilizer_C.

TABLE 10 PEP SEQ Pfam domain ID NO GENE ID name begin stop score E-value194 PHE0003351_PMON81242.pep PPDK_N 99 464 710.9 7.90E−211 194PHE0003351_PMON81242.pep PEP-utilizers 500 601 182.3 1.10E−51 194PHE0003351_PMON81242.pep PEP-utilizers_C 613 969 723.9 1.00E−214 195PHE0003351_PMON83625.pep PPDK_N 99 464 710.9 7.90E−211 195PHE0003351_PMON83625.pep PEP-utilizers 500 601 182.3 1.10E−51 195PHE0003351_PMON83625.pep PEP-utilizers_C 613 969 723.9 1.00E−214 196PHE0000207_PMON77878.pep Pkinase 1 259 343.1 4.40E−100 197PHE0000208_PMON77879.pep Pkinase 1 259 353.4 3.30E−103 198PHE0000209_PMON77891.pep Pkinase 1 259 354.9 1.20E−103 199PHE0000210_PMON77880.pep Pkinase 1 259 359.4 5.40E−105 200PHE0001329_PMON92878.pep Pkinase 12 266 354.3 1.80E−103 200PHE0001329_PMON92878.pep NAF 311 371 123.6 5.10E−34 201PHE0001425_PMON79162.pep CAF1 19 252 368.1 1.30E−107 202PHE0001573_PMON92870.pep GATase_2 2 162 55.5 3.10E−15 202PHE0001573_PMON92870.pep Asn_synthase 210 451 329.6 5.00E−96 203PHE0001664_PMON99280.pep FAD_binding_4 69 213 83.4 6.30E−22 204PHE0001674_PMON79194.pep Myb_DNA-binding 25 70 36.3 9.90E−08 205PHE0002026_PMON96489.pep Ammonium_transp 36 459 628.5 5.10E−186 206PHE0002108_PMON92821.pep CSD 1 65 155.1 1.60E−43 207PHE0002109_PMON93856.pep CSD 1 67 144.8 2.10E−40 208PHE0002508_PMON92607.pep CBFD_NFYB_HMF 24 89 130.9 3.20E−36 209PHE0002650_PMON81832.pep SRF-TF 9 59 106.9 5.50E−29 209PHE0002650_PMON81832.pep K-box 73 172 118.4 1.90E−32 210PHE0002989_PMON95630.pep Miro 10 126 74.3 3.40E−19 210PHE0002989_PMON95630.pep Ras 11 173 288.8 9.30E−84 212PHE0003300_PMON95106.pep MtN3_slv 12 99 131.1 2.80E−36 212PHE0003300_PMON95106.pep MtN3_slv 133 219 134.9 2.00E−37 214PHE0003389_PMON94682.pep p450 48 527 286.5 4.60E−83 215PHE0003614_PMON95111.pep Pyridoxal_deC 33 381 531.8 6.80E−157 216PHE0003684_PMON92807.pep Myb_DNA-binding 118 168 47.9 3.00E−11 217PHE0003684_PMON93378.pep Myb_DNA-binding 118 168 47.9 3.00E−11 218PHE0003853_PMON92602.pep Cyclin_N 46 171 72.6 1.10E−18 219PHE0003903_PMON98271.pep TPP_enzyme_N 44 220 302.9 5.50E−88 219PHE0003903_PMON98271.pep TPP_enzyme_M 241 390 157.3 3.70E−44 220PHE0003905_PMON99283.pep Aldedh 30 492 514.4 1.10E−151 221PHE0003907_PMON98066.pep Ribosomal_L12 124 191 62.6 1.20E−15 222PHE0003908_PMON98064.pep DnaJ 31 93 128.9 1.30E−35 223PHE0003960_PMON95079.pep CTP_transf_2 56 186 142.9 7.70E−40 224PHE0003967_PMON95088.pep GST_N 11 84 43.3 7.40E−10 228PHE0004023_PMON92446.pep PHD 198 248 54.9 2.40E−13 229PHE0004026_PMON93885.pep Aa_trans 44 438 409.4 4.80E−120 230PHE0004027_PMON93860.pep FAD_binding_4 64 218 83.9 4.60E−22 231PHE0004028_PMON94697.pep Alpha-amylase 10 426 −62.1 4.30E−06 232PHE0004034_PMON92631.pep DUF1336 236 478 491.8 7.30E−145 234PHE0004047_PMON92619.pep LIM 11 68 53.4 7.00E−13 234PHE0004047_PMON92619.pep LIM 110 167 63.9 4.70E−16 235PHE0004047_PMON93388.pep LIM 11 68 53.4 7.00E−13 235PHE0004047_PMON93388.pep LIM 110 167 63.9 4.70E−16 236PHE0004068_PMON93663.pep AWPM-19 1 125 287.6 2.20E−83 237PHE0004071_PMON93311.pep RRM_1 105 174 77.3 4.40E−20 238PHE0004072_PMON93654.pep MMR_HSR1 214 324 65.7 1.40E−16 239PHE0004072_PMON93669.pep MMR_HSR1 214 324 65.7 1.40E−16 241PHE0004075_PMON92851.pep MtN3_slv 15 104 75.5 1.50E−19 241PHE0004075_PMON92851.pep MtN3_slv 137 223 97.2 4.50E−26 242PHE0004080_PMON93321.pep peroxidase 19 227 241 2.40E−69 243PHE0004084_PMON95141.pep Phi_1 35 314 691.3 6.40E−205 244PHE0004093_PMON93332.pep Dimerisation 40 100 105.7 1.20E−28 244PHE0004093_PMON93332.pep Methyltransf_2 104 350 317.5 2.20E−92 245PHE0004093_PMON94155.pep Dimerisation 40 100 105.7 1.20E−28 245PHE0004093_PMON94155.pep Methyltransf_2 104 350 317.5 2.20E−92 247PHE0004144_PMON93842.pep Cofilin_ADF 16 143 152.4 1.10E−42 248PHE0004148_PMON92574.pep Iso_dh 28 412 521.5 8.40E−154 249PHE0004149_PMON92471.pep HEAT_PBS 115 143 23.8 0.00057 249PHE0004149_PMON92471.pep HEAT_PBS 155 181 37.8 3.50E−08 249PHE0004149_PMON92471.pep HEAT_PBS 186 212 22.4 0.0015 249PHE0004149_PMON92471.pep HEAT_PBS 260 287 16.8 0.074 250PHE0004149_PMON93899.pep HEAT_PBS 115 143 23.8 0.00057 250PHE0004149_PMON93899.pep HEAT_PBS 155 181 37.8 3.50E−08 250PHE0004149_PMON93899.pep HEAT_PBS 186 212 22.4 0.0015 250PHE0004149_PMON93899.pep HEAT_PBS 260 287 16.8 0.074 251PHE0004152_PMON93672.pep AT_hook 69 81 7.4 1.1 251PHE0004152_PMON93672.pep DUF296 96 217 175.1 1.60E−49 252PHE0004155_PMON92626.pep ADH_N 41 152 176.1 8.20E−50 252PHE0004155_PMON92626.pep ADH_zinc_N 181 324 127.8 2.70E−35 253PHE0004156_PMON92623.pep NPH3 135 364 219.3 7.80E−63 254PHE0004162_PMON92481.pep AUX_IAA 22 279 395.9 5.30E−116 255PHE0004164_PMON92465.pep X8 29 115 168.4 1.70E−47 257PHE0004167_PMON93333.pep APS_kinase 108 264 363.4 3.20E−106 258PHE0004168_PMON93855.pep FAD_binding_4 84 225 93 8.10E−25 258PHE0004168_PMON93855.pep BBE 476 534 120.1 5.70E−33 259PHE0004169_PMON92568.pep Aldo_ket_red 14 298 389.4 4.80E−114 260PHE0004184_PMON92565.pep UIM 214 231 14.4 0.29 260PHE0004184_PMON92565.pep UIM 298 315 24 0.00049 261PHE0004185_PMON92802.pep p450 39 504 157.8 2.70E−44 262PHE0004188_PMON92803.pep HSF_DNA-bind 70 233 177.8 2.50E−50 263PHE0004190_PMON92801.pep HLH 175 213 9.2 0.014 264PHE0004208_PMON92834.pep Myb_DNA-binding 5 56 39.1 1.30E−08 264PHE0004208_PMON92834.pep Myb_DNA-binding 134 181 44.6 3.10E−10 265PHE0004215_PMON92827.pep PBP 14 170 30 1.50E−07 266PHE0004223_PMON92840.pep Fasciclin 40 179 11.7 0.00032 266PHE0004223_PMON92840.pep Fasciclin 217 353 104.9 2.20E−28 267PHE0004225_PMON94167.pep Aldedh 77 539 880.7 6.30E−262 268PHE0004226_PMON95114.pep Aldedh 77 539 873 1.30E−259 269PHE0004227_PMON92605.pep UPF0057 5 55 76.3 8.50E−20 270PHE0004229_PMON92867.pep UPF0057 4 54 96.4 8.00E−26 271PHE0004233_PMON92843.pep HSF_DNA-bind 60 236 255.5 1.00E−73 272PHE0004237_PMON93673.pep HSP20 48 153 184.3 2.80E−52 273PHE0004243_PMON92621.pep CBFD_NFYB_HMF 22 87 122.5 1.10E−33 274PHE0004244_PMON92858.pep CBFD_NFYB_HMF 39 104 121 3.00E−33 275PHE0004245_PMON93813.pep CBFD_NFYB_HMF 25 90 129.2 1.00E−35 276PHE0004248_PMON94672.pep CBFD_NFYB_HMF 37 102 125.3 1.60E−34 278PHE0004250_PMON92881.pep CBFD_NFYB_HMF 25 90 119.7 7.80E−33 279PHE0004252_PMON92606.pep CBFD_NFYB_HMF 14 79 94.1 3.80E−25 280PHE0004253_PMON92874.pep CBFD_NFYB_HMF 7 71 83.8 5.00E−22 281PHE0004258_PMON93385.pep Pkinase 5 276 144.7 2.30E−40 282PHE0004258_PMON93806.pep Pkinase 5 276 144.7 2.30E−40 283PHE0004259_PMON93384.pep Abhydrolase_3 95 319 302.4 7.70E−88 285PHE0004261_PMON93389.pep Pkinase 31 282 289.6 5.30E−84 285PHE0004261_PMON93389.pep Pkinase_Tyr 31 280 69.3 6.10E−20 286PHE0004261_PMON93655.pep Pkinase 31 282 289.6 5.30E−84 286PHE0004261_PMON93655.pep Pkinase_Tyr 31 280 69.3 6.10E−20 287PHE0004262_PMON92862.pep Pkinase 86 366 153.9 3.70E−43 287PHE0004262_PMON92862.pep Pkinase_Tyr 86 366 132.2 1.30E−36 288PHE0004262_PMON93360.pep Pkinase 86 366 153.9 3.70E−43 288PHE0004262_PMON93360.pep Pkinase_Tyr 86 366 132.2 1.30E−36 289PHE0004264_PMON92845.pep PMEI 25 174 138.8 1.40E−38 290PHE0004264_PMON93354.pep PMEI 25 174 138.8 1.40E−38 291PHE0004265_PMON92873.pep Suc_Fer-like 59 308 60.7 4.30E−15 292PHE0004265_PMON93807.pep Suc_Fer-like 59 308 60.7 4.30E−15 293PHE0004266_PMON92877.pep Myb_DNA-binding 298 348 46.6 7.80E−11 294PHE0004284_PMON93857.pep U-box 23 97 98.2 2.30E−26 295PHE0004285_PMON95136.pep CBFD_NFYB_HMF 61 126 123 7.70E−34 296PHE0004286_PMON93666.pep ICL 21 551 1239.3 0 297PHE0004287_PMON93344.pep ICL 21 552 1169.2 0 298PHE0004307_PMON94102.pep RWP-RK 196 247 90.7 4.00E−24 299PHE0004314_PMON93397.pep zf-C3HC4 148 185 34.1 4.50E−07 300PHE0004321_PMON93811.pep Redoxin 64 228 4.9 0.0016 300PHE0004321_PMON93811.pep GSHPx 73 181 246.5 5.10E−71 301PHE0004321_PMON93834.pep Redoxin 64 228 4.9 0.0016 301PHE0004321_PMON93834.pep GSHPx 73 181 246.5 5.10E−71 302PHE0004325_PMON93818.pep CcmH 1 139 16.6 6.50E−09 303PHE0004335_PMON93850.pep DZC 158 193 81.9 1.80E−21 303PHE0004335_PMON93850.pep DZC 308 343 80.9 3.70E−21 304PHE0004336_PMON93858.pep DZC 179 214 78.3 2.20E−20 304PHE0004336_PMON93858.pep DZC 369 404 71.4 2.60E−18 306PHE0004348_PMON93810.pep CSD 1 65 136.8 5.50E−38 307PHE0004349_PMON93812.pep CSD 1 65 141.9 1.50E−39 308PHE0004350_PMON93826.pep CSD 1 66 148.4 1.70E−41 309PHE0004351_PMON93821.pep CSD 1 66 149.5 8.10E−42 310PHE0004352_PMON93824.pep CSD 2 68 151.2 2.50E−42 312PHE0004393_PMON94192.pep efhand 29 57 18 0.031 312PHE0004393_PMON94192.pep efhand 66 94 25.3 0.0002 312PHE0004393_PMON94192.pep efhand 110 138 24.2 0.00042 313PHE0004395_PMON94145.pep C2 16 138 72.1 1.60E−18 313PHE0004395_PMON94145.pep PLDc 357 392 30.1 7.20E−06 313PHE0004395_PMON94145.pep PLDc 702 729 37.1 5.70E−08 314PHE0004396_PMON94137.pep Orn_Arg_deC_N 118 393 282.3 8.80E−82 314PHE0004396_PMON94137.pep Orn_DAP_Arg_deC 396 596 161.7 1.70E−45 315PHE0004417_PMON94190.pep Spermine_synth 13 256 516.1 3.40E−152 316PHE0004418_PMON94368.pep Amino_oxidase 18 504 275.8 7.90E−80 317PHE0004419_PMON95100.pep Amidohydro_1 95 446 56.2 1.00E−13 317PHE0004419_PMON95100.pep Amidohydro_3 95 444 −49.9 0.00024 318PHE0004421_PMON95120.pep AP2 52 118 92.2 1.40E−24 319PHE0004422_PMON95123.pep AP2 58 123 78.5 1.90E−20 322PHE0004432_PMON94112.pep Lactamase_B 63 262 81.6 2.20E−21 322PHE0004432_PMON94112.pep RMMBL 400 440 33.6 6.10E−07 323PHE0004472_PMON94115.pep Sina 5 205 188 2.00E−53 324PHE0004472_PMON94126.pep Sina 5 205 188 2.00E−53 325PHE0004488_PMON95609.pep Anti-silence 1 155 392.9 4.40E−115 327PHE0004492_PMON95614.pep NPH3 193 435 469.9 2.90E−138 328PHE0004545_PMON95117.pep Ribosomal_L14 49 196 105.5 1.50E−28 329PHE0004574_PMON94433.pep Transaldolase 102 405 620.7 1.20E−183 329PHE0004574_PMON94433.pep efhand 444 472 21.3 0.0031 330PHE0004606_PMON95627.pep Metallophos 54 249 154 3.60E−43 331PHE0004620_PMON94189.pep PFK 6 281 515.1 7.30E−152 332PHE0004620_PMON94442.pep PFK 6 281 515.1 7.30E−152 333PHE0004622_PMON95621.pep F-box 2 49 43.2 8.10E−10 333PHE0004622_PMON95621.pep LRR_2 150 175 41.5 2.60E−09 333PHE0004622_PMON95621.pep FBD 332 382 74.8 2.50E−19 334PHE0004626_PMON95101.pep Aminotran_3 79 434 323.4 3.50E−94 335PHE0004630_PMON94367.pep Iso_dh 40 363 326.9 3.20E−95 336PHE0004634_PMON94385.pep AP2 28 91 114.2 3.40E−31 337PHE0004640_PMON95066.pep FAE1_CUT1_RppA 75 365 539.9 2.40E−159 337PHE0004640_PMON95066.pep Chal_sti_synt_C 322 466 8.7 0.0003 337PHE0004640_PMON95066.pep ACP_syn_III_C 382 464 26.7 2.30E−08 338PHE0004645_PMON94655.pep 14-3-3 5 241 304.8 1.50E−88 339PHE0004645_PMON94685.pep 14-3-3 5 241 304.8 1.50E−88 342PHE0004650_PMON94686.pep Skp1_POZ 4 64 105.3 1.70E−28 342PHE0004650_PMON94686.pep Skp1 112 190 173 6.90E−49 343PHE0004652_PMON94657.pep UPF0005 31 247 55.1 2.20E−13 344PHE0004652_PMON94687.pep UPF0005 31 247 55.1 2.20E−13 346PHE0004689_PMON95131.pep Pkinase 12 291 357 2.70E−104 347PHE0004691_PMON95129.pep Spermine_synth 33 278 501.4 9.60E−148 348PHE0004719_PMON94698.pep zf-C3HC4 203 243 26.6 8.00E−05 349PHE0004719_PMON95089.pep zf-C3HC4 203 243 26.6 8.00E−05 350PHE0004734_PMON94667.pep KOW 26 62 30.7 4.80E−06 350PHE0004734_PMON94667.pep eIF-5a 84 153 125.8 1.10E−34 351PHE0004735_PMON95116.pep KOW 26 62 32.2 1.70E−06 351PHE0004735_PMON95116.pep eIF-5a 84 153 120.3 5.10E−33 352PHE0004739_PMON95110.pep Miro 7 121 68.7 1.70E−17 352PHE0004739_PMON95110.pep Ras 8 179 270.9 2.30E−78 353PHE0004753_PMON95105.pep Aldedh 61 520 791.8 3.50E−235 355PHE0004770_PMON95122.pep DUF1242 2 70 118.4 1.80E−32 357PHE0004774_PMON95147.pep zf-A20 14 38 33.1 9.10E−07 357PHE0004774_PMON95147.pep zf-AN1 92 132 68.2 2.50E−17 358PHE0004777_PMON95118.pep RNA_pol_L 6 83 75.9 1.20E−19 359PHE0004785_PMON95057.pep Ribosomal_L18p 26 172 251.4 1.70E−72 360PHE0004786_PMON95604.pep Phi_1 35 314 691.3 6.40E−205 361PHE0004788_PMON95092.pep DS 53 369 587.4 1.30E−173 362PHE0004799_PMON95602.pep DAO 34 481 −14.1 7.30E−05 362PHE0004799_PMON95602.pep Amino_oxidase 42 483 342.7 5.60E−100 363PHE0004841_PMON95636.pep DNA_photolyase 18 190 254.3 2.30E−73 363PHE0004841_PMON95636.pep FAD_binding_7 223 501 503 3.20E−148 367PHE0004888_PMON95603.pep Globin 7 134 73 8.40E−19 367PHE0004888_PMON95603.pep FAD_binding_6 156 263 29 4.80E−07 367PHE0004888_PMON95603.pep NAD_binding_1 276 393 13.4 9.80E−05 369ERD4.pep DUF221 295 710 245.3 1.20E−70 370 At1g78070.2.pep WD40 310 34734.1 4.40E−07 372 At3g47340.1.pep GATase_2 2 161 99.6 8.60E−27 372At3g47340.1.pep Asn_synthase 209 450 344.3 1.80E−100 373 At3g47340.3.pepGATase_2 2 161 99.6 8.60E−27 373 At3g47340.3.pep Asn_synthase 209 430286.1 6.20E−83 374 At3g47340.2.pep GATase_2 2 161 99.6 8.60E−27 374At3g47340.2.pep Asn_synthase 209 450 344.3 1.80E−100 375 At5g13170.1.pepMtN3_slv 12 99 135.1 1.80E−37 375 At5g13170.1.pep MtN3_slv 134 220 135.41.40E−37 376 At2g19900.1.pep malic 107 295 407.1 2.20E−119 376At2g19900.1.pep Malic_M 297 550 466.9 2.30E−137 379 At2g42790.1.pepCitrate_synt 93 461 506.2 3.40E−149 380 At3g56200.1.pep Aa_trans 21 426106.3 8.10E−29 381 At5g01520.1.pep zf-C3HC4 146 183 26.1 0.00011 384At5g59320.1.pep Tryp_alpha_amyl 27 111 114.7 2.30E−31 385 AtHB7.pepHomeobox 30 86 66.6 7.10E−17 385 AtHB7.pep HALZ 87 131 39.2 1.30E−08 386RD20.pep Caleosin 54 227 469 5.30E−138

TABLE 11 Pfam domain accession gathering name number cutoff domaindescription 14-3-3 PF00244.9 25 14-3-3 protein ACP_syn_III_C PF08541.1−24.4 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III C terminalADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain ADH_zinc_NPF00107.16 23.8 Zinc-binding dehydrogenase AP2 PF00847.9 0 AP2 domainAPS_kinase PF01583.9 25 Adenylylsulphate kinase AT_hook PF02178.8 3.6 AThook motif AUX_IAA PF02309.6 −83 AUX/IAA family AWPM-19 PF05512.1 25AWPM-19-like family Aa_trans PF01490.7 −128.4 Transmembrane amino acidtransporter protein Abhydrolase_3 PF07859.2 25.8 alpha/beta hydrolasefold Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family Aldo_ket_redPF00248.10 −97 Aldo/keto reductase family Alpha-amylase PF00128.12 −93Alpha amylase, catalytic domain Amidohydro_1 PF01979.8 −37.4Amidohydrolase family Amidohydro_3 PF07969.1 −65.5 Amidohydrolase familyAmino_oxidase PF01593.12 −11.4 Flavin containing amine oxidoreductaseAminotran_3 PF00202.10 −207.6 Aminotransferase class-III Ammonium_transpPF00909.10 −144 Ammonium Transporter Family Anti-silence PF04729.4 25Anti-silencing protein, ASF1-like Asn_synthase PF00733.10 −52.8Asparagine synthase BBE PF08031.1 25 Berberine and berberine like C2PF00168.18 3.7 C2 domain CAF1 PF04857.8 −100.5 CAF1 family ribonucleaseCBFD_NFYB_HMF PF00808.12 18.4 Histone-like transcription factor(CBF/NF-Y) and archaeal histone CSD PF00313.12 −0.3 ‘Cold-shock’DNA-binding domain CTP_transf_2 PF01467.16 −11.8 CytidylyltransferaseCaleosin PF05042.3 25 Caleosin related protein CcmH PF03918.4 −30.8Cytochrome C biogenesis protein Chal_sti_synt_C PF02797.5 −6.1 Chalconeand stilbene synthases, C-terminal domain Citrate_synt PF00285.10 −101.5Citrate synthase Cofilin_ADF PF00241.10 −4.7 Cofilin/tropomyosin-typeactin-binding protein Cyclin_N PF00134.13 −14.7 Cyclin, N-terminaldomain DAO PF01266.12 −35.9 FAD dependent oxidoreductase DNA_photolyasePF00875.8 26.1 DNA photolyase DS PF01916.7 −95.2 Deoxyhypusine synthaseDUF1242 PF06842.1 25 Protein of unknown function (DUF1242) DUF1336PF07059.2 −78.2 Protein of unknown function (DUF1336) DUF221 PF02714.525 Domain of unknown function DUF221 DUF296 PF03479.4 −11 Domain ofunknown function (DUF296) DZC PF08381.1 15.3 Disease resistance/zincfinger/chromosome condensation-like region Dimerisation PF08100.1 18.1Dimerisation domain DnaJ PF00226.19 −8 DnaJ domain F-box PF00646.21 13.6F-box domain FAD_binding_4 PF01565.12 −8.1 FAD binding domainFAD_binding_6 PF00970.13 −11.4 Oxidoreductase FAD-binding domainFAD_binding_7 PF03441.4 25 FAD binding domain of DNA photolyaseFAE1_CUT1_RppA PF08392.1 −192.7 FAE1/Type III polyketide synthase-likeprotein FBD PF08387.1 25 FBD Fasciclin PF02469.10 4 Fasciclin domainGATase_2 PF00310.10 −106.2 Glutamine amidotransferases class-II GSHPxPF00255.10 −16 Glutathione peroxidase GST_N PF02798.9 14.6 GlutathioneS-transferase, N-terminal domain Globin PF00042.11 −8.8 Globin HALZPF02183.7 17 Homeobox associated leucine zipper HEAT_PBS PF03130.5 15PBS lyase HEAT-like repeat HLH PF00010.15 8.2 Helix-loop-helixDNA-binding domain HSF_DNA-bind PF00447.7 −70 HSF-type DNA-binding HSP20PF00011.10 13 Hsp20/alpha crystallin family Homeobox PF00046.18 −4.1Homeobox domain ICL PF00463.10 −234 Isocitrate lyase family Iso_dhPF00180.10 −97 Isocitrate/isopropylmalate dehydrogenase K-box PF01486.70 K-box region KOW PF00467.18 29.1 KOW motif LIM PF00412.11 0 LIM domainLRR_2 PF07723.2 6 Leucine Rich Repeat Lactamase_B PF00753.16 24.6Metallo-beta-lactamase superfamily MMR_HSR1 PF01926.11 31.2 GTPase ofunknown function Malic_M PF03949.4 −143.9 Malic enzyme, NAD bindingdomain Metallophos PF00149.17 22 Calcineurin-like phosphoesteraseMethyltransf_2 PF00891.7 −103.8 O-methyltransferase Miro PF08477.1 28Miro-like protein MtN3_slv PF03083.5 −0.8 MtN3/saliva familyMyb_DNA-binding PF00249.19 2.8 Myb-like DNA-binding domain NAD_binding_1PF00175.10 −3.9 Oxidoreductase NAD-binding domain NAF PF03822.4 4.5 NAFdomain NPH3 PF03000.4 25 NPH3 family Orn_Arg_deC_N PF02784.7 −76Pyridoxal-dependent decarboxylase, pyridoxal binding domainOrn_DAP_Arg_deC PF00278.12 6.7 Pyridoxal-dependent decarboxylase,C-terminal sheet domain PBP PF01161.9 −20.6Phosphatidylethanolamine-binding protein PEP-utilizers PF00391.12 10PEP-utilising enzyme, mobile domain PEP-utilizers_C PF02896.7 −173PEP-utilising enzyme, TIM barrel domain PFK PF00365.10 −132Phosphofructokinase PHD PF00628.17 25.9 PHD-finger PLDc PF00614.11 0Phospholipase D Active site motif PMEI PF04043.5 25 Plantinvertase/pectin methylesterase inhibitor PPDK_N PF01326.8 −87 Pyruvatephosphate dikinase, PEP/pyruvate binding domain Phi_1 PF04674.2 25Phosphate-induced protein 1 conserved region Pkinase PF00069.14 −70.8Protein kinase domain Pkinase_Tyr PF07714.5 65 Protein tyrosine kinasePyridoxal_deC PF00282.9 −158.6 Pyridoxal-dependent decarboxylaseconserved domain RMMBL PF07521.1 18.5 RNA-metabolisingmetallo-beta-lactamase RNA_pol_L PF01193.12 16.9 RNA polymeraseRpb3/Rpb11 dimerisation domain RRM_1 PF00076.11 20.7 RNA recognitionmotif. (a.k.a. RRM, RBD, or RNP domain) RWP-RK PF02042.5 25 RWP-RKdomain Ras PF00071.11 −69.9 Ras family Redoxin PF08534.1 −1 RedoxinRibosomal_L12 PF00542.8 25 Ribosomal protein L7/L12 C-terminal domainRibosomal_L14 PF00238.9 −8 Ribosomal protein L14p/L23e Ribosomal_L18pPF00861.12 25 Ribosomal L18p/L5e family SRF-TF PF00319.8 11 SRF-typetranscription factor (DNA-binding and dimerisation domain) SinaPF03145.6 −48.4 Seven in absentia protein family Skp1 PF01466.8 −2 Skp1family, dimerisation domain Skp1_POZ PF03931.5 14.9 Skp1 family,tetramerisation domain Spermine_synth PF01564.6 −93.8Spermine/spermidine synthase Suc_Fer-like PF06999.2 −42.4Sucrase/ferredoxin-like TPP_enzyme_M PF00205.11 −23.9 Thiaminepyrophosphate enzyme, central domain TPP_enzyme_N PF02776.7 −70 Thiaminepyrophosphate enzyme, N-terminal TPP binding domain TransaldolasePF00923.9 −49 Transaldolase Tryp_alpha_amyl PF00234.10 −4 Proteaseinhibitor/seed storage/LTP family U-box PF04564.5 10.5 U-box domain UIMPF02809.10 4.1 Ubiquitin interaction motif UPF0005 PF01027.11 −6.7Uncharacterised protein family UPF0005 UPF0057 PF01679.7 25Uncharacterized protein family UPF0057 WD40 PF00400.20 21.5 WD domain,G-beta repeat X8 PF07983.3 −28.8 X8 domain eIF-5a PF01287.9 9.6Eukaryotic initiation factor 5A hypusine, DNA- binding OB fold efhandPF00036.20 17.5 EF hand malic PF00390.8 25 Malic enzyme, N-terminaldomain p450 PF00067.11 −105 Cytochrome P450 peroxidase PF00141.12 −10Peroxidase zf-A20 PF01754.6 25 A20-like zinc finger zf-AN1 PF01428.6 0AN1-like Zinc finger zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type(RING finger)

Example 10 Selection of Transgenic Plants with Enhanced Agronomic Traits

This example illustrates identification of plant cells of the inventionby screening transgenic plants and seeds for an enhanced trait.Transgenic seed and plants, e.g., with transgenic corn cells in theplants prepared in Example 2, transgenic soybean cells in the plantsprepared in Example 3, transgenic cotton cells in the plants prepared inExample 4, and transgenic cells in the plants prepared in Example 6, arescreened for enhanced water use efficiency, enhanced cold tolerance,increased yield, enhanced nitrogen use efficiency, enhanced seed proteinand enhanced seed oil as compared to control plants.

A. Selection for Enhanced Nitrogen Use Efficiency

The physiological efficacy of transgenic corn plants (tested as hybrids)can be tested for nitrogen use efficiency (NUE) traits in ahigh-throughput nitrogen (N) selection method. The collected data arecompared to the measurements from wildtype controls using a statisticalmodel to determine if the changes are due to the transgene. Raw datawere analyzed by SAS software. Results shown herein are the comparisonof transgenic plants relative to the wildtype controls.

(1) Media Preparation for Planting a NUE Protocol

Planting materials used: Metro Mix 200 (vendor: Hummert) Cat. #10-0325,Scotts Micro Max Nutrients (vendor: Hummert) Cat. #07-6330, OS 4⅓″×3⅞″pots (vendor: Hummert) Cat. #16-1415, OS trays (vendor: Hummert) Cat.#16-1515, Hoagland's macronutrients solution, Plastic 5″ stakes (vendor:Hummert) yellow Cat. #49-1569, white Cat. #49-1505, Labels with numbersindicating material contained in pots. Fill 500 pots to rim with MetroMix 200 to a weight of ˜140 g/pot. Pots are filled uniformly by using abalancer. Add 0.4 g of Micro Max nutrients to each pot. Stir ingredientswith spatula to a depth of 3 inches while preventing material loss.

(2) Planting a NUE Selection in the Greenhouse

(a) Seed Germination—Each pot is lightly atered twice using reverseosmosis purified water. The first watering is scheduled to occur justbefore planting; and the second watering, after the seed has beenplanted in the pot. Ten Seeds of each entry (1 seed per pot) are plantedto select eight healthy uniform seedlings. Additional wild type controlsare planted for use as border rows. Alternatively, 15 seeds of eachentry (1 seed per pot) are planted to select 12 healthy uniformseedlings (this larger number of plantings is used for the second, orconfirmation, planting). Place pots on each of the 12 shelves in theConviron growth chamber for seven days. This is done to allow moreuniform germination and early seedling growth. The following growthchamber settings are 25° C./day and 22° C./night, 14 hours light and tenhours dark, humidity ˜80%, and light intensity ˜350 μmol/m²/s (at potlevel). Watering is done via capillary matting similar to greenhousebenches with duration of ten minutes three times a day.

(b) Seedling transfer—After seven days, the best eight or 12 seedlingsfor the first or confirmation pass runs, respectively, are chosen andtransferred to greenhouse benches. The pots are spaced eight inchesapart (center to center) and are positioned on the benches using thespacing patterns printed on the capillary matting. The Vattex mattingcreates a 384-position grid, randomizing all range, row combinations.Additional pots of controls are placed along the outside of theexperimental block to reduce border effects.

Plants are allowed to grow for 28 days under the low N run or for 23days under the high N run. The macronutrients are dispensed in the formof a macronutrient solution (see composition below) containing preciseamounts of N added (2 mM NH₄NO₃ for limiting N selection and 20 mMNH₄NO₃ for high N selection runs). Each pot is manually dispensed 100 mlof nutrient solution three times a week on alternate days starting ateight and ten days after planting for high N and low N runs,respectively. On the day of nutrient application, two 20 min wateringsat 05:00 and 13:00 are skipped. The vattex matting should be changedevery third run to avoid N accumulation and buildup of root matter.Table 12 shows the amount of nutrients in the nutrient solution foreither the low or high nitrogen selection.

TABLE 12 2 mM NH₄NO₃ 20 mM NH₄NO₃ (Low Nitrogen (high Nitrogen GrowthCondition) Growth Condition) Nutrient Stock mL/L mL/L 1 M NH₄N0₃ 2 20 1M KH₂PO₄ 0.5 0.5 1 M MgSO₄ · 7H₂O 2 2 1 M CaCl₂ 2.5 2.5 1 M K₂SO₄ 1 1Note: Adjust pH to 5.6 with HCl or KOH

(c) Harvest Measurements and Data Collection—After 28 days of plantgrowth for low N runs and 23 days of plant growth for high N runs, thefollowing measurements are taken (phenocodes in parentheses): totalshoot fresh mass (g) (SFM) measured by Sartorius electronic balance, V6leaf chlorophyll measured by Minolta SPAD meter (relative units) (LC),V6 leaf area (cm²) (LA) measured by a Li-Cor leaf area meter, V6 leaffresh mass (g) (LFM) measured by Sartorius electronic balance, and V6leaf dry mass (g) (LDM) measured by Sartorius electronic balance. Rawdata were analyzed by SAS software. Results shown are the comparison oftransgenic plants relative to the wildtype controls.

To take a leaf reading, samples were excised from the V6 leaf. Sincechlorophyll meter readings of corn leaves are affected by the part ofthe leaf and the position of the leaf on the plant that is sampled, SPADmeter readings were done on leaf six of the plants. Three measurementsper leaf were taken, of which the first reading was taken from a pointone-half the distance between the leaf tip and the collar and halfwayfrom the leaf margin to the midrib while two were taken toward the leaftip. The measurements were restricted in the area from ½ to ¾ of thetotal length of the leaf (from the base) with approximately equalspacing between them. The average of the three measurements was takenfrom the SPAD machine.

Leaf fresh mass is recorded for an excised V6 leaf, the leaf is placedinto a paper bag. The paper bags containing the leaves are then placedinto a forced air oven at 80° C. for 3 days. After 3 days, the paperbags are removed from the oven and the leaf dry mass measurements aretaken.

From the collected data, two derived measurements are made: (1) Leafchlorophyll area (LCA), which is a product of V6 relative chlorophyllcontent and its leaf area (relative units). Leaf chlorophyll area=leafchlorophyll X leaf area. This parameter gives an indication of thespread of chlorophyll over the entire leaf area; (2) specific leaf area(LSA) is calculated as the ratio of V6 leaf area to its dry mass (cm²/gdry mass), a parameter also recognized as a measure of NUE.

Nitrogen Use Field Efficacy Assay

Level I. Transgenic plants provided by the present invention are plantedin field without any nitrogen source being applied. Transgenic plantsand control plants are grouped by genotype and construct with controlsarranged randomly within genotype blocks. Each type of transgenic plantsare tested by 3 replications and across 5 locations. Nitrogen levels inthe fields are analyzed in early April pre-planting by collecting 30sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples areanalyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organicmatter and pH to provide baseline values. P, K and micronutrients areapplied based upon soil test recommendations.

Level II. Transgenic plants provided by the present invention areplanted in field with three levels of nitrogen (N) fertilizer beingapplied, i.e. low level (0 N), medium level (80 lb/ac) and high level(180 lb/ac). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used asthe N source and apply by broadcast boom and incorporate with a fieldcultivator with rear rolling basket in the same direction as intendedcrop rows. Although there is no N applied to the 0 N treatment the soilshould still be disturbed in the same fashion as the treated area.Transgenic plants and control plants are grouped by genotype andconstruct with controls arranged randomly within genotype blocks. Eachtype of transgenic plants is tested by 3 replications and across 4locations. Nitrogen levels in the fields are analyzed in early Aprilpre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″soil layer. Soil samples are analyzed for nitrate-nitrogen,phosphorus(P), Potassium(K), organic matter and pH to provide baselinevalues. P, K and micronutrients are applied based upon soil testrecommendations.

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield ascompared to a control plant. Improved yield can result from enhancedseed sink potential, i.e. the number and size of endosperm cells orkernels and/or enhanced sink strength, i.e. the rate of starchbiosynthesis. Sink potential can be established very early during kerneldevelopment, as endosperm cell number and size are determined within thefirst few days after pollination.

Effective yield selection of enhanced yielding transgenic corn eventsuses hybrid progeny of the transgenic event over multiple locations withplants grown under optimal production management practices, and maximumpest control. A useful target for improved yield is a 5% to 10% increasein yield as compared to yield produced by plants grown from seed for acontrol plant. Selection methods may be applied in multiple and diversegeographic locations, for example up to 16 or more locations, over oneor more plating seasons, for example at least two planting seasons tostatistically distinguish yield improvement from natural environmentaleffects. It is to plant multiple transgenic plants, positive andnegative control plants, and pollinator plants in standard plots, forexample 2 row plots, 20 feet long by 5 feet wide with 30 inches distancebetween rows and a 3 foot alley between ranges. Transgenic events can begrouped by recombinant DNA constructs with groups randomly placed in thefield. A pollinator plot of a high quality corn line is planted forevery two plots to allow open pollination when using male steriletransgenic events. A useful planting density is about 30,000plants/acre. High planting density is greater than 30,000 plants/acre,preferably about 40,000 plants/acre, more preferably about 42,000plants/acre, most preferably about 45,000 plants/acre. Surrogateindicators for yield improvement include source capacity (biomass),source output (sucrose and photosynthesis), sink components (kernelsize, ear size, starch in the seed), development (light response,height, density tolerance), maturity, early flowering trait andphysiological responses to high density planting, for example at 45,000plants per acre, for example as illustrated in Table 13 and 14.

TABLE 13 Timing Evaluation Description comments V2-3 Early stand Can betaken any time after germination and prior to removal of any plants.Pollen GDU to 50% GDU to 50% plants shedding shed shed 50% tassel.Silking GDU to 50% GDU to 50% plants showing silk silks. Maturity Plantheight Height from soil surface to 10 plants per plot- flag leafattachment (inches). Yield team assistance Maturity Ear height Heightfrom soil surface to 10 plants per plot- primary ear attachment node.Yield team assistance Maturity Leaves above visual scores: erect, size,ear rolling Maturity Tassel size Visual scores +/− vs. WT Pre- FinalStand Final stand count prior to Harvest harvest, exclude tillers Pre-Stalk lodging No. of stalks broken below Harvest the primary earattachment. Exclude leaning tillers Pre- Root lodging No. of stalksleaning >45° Harvest angle from perpendicular. Pre- Stay green Afterphysiological maturity Harvest and when differences among genotypes areevident: Scale 1 (90-100% tissue green)-9 (0-19% tissue green). HarvestGrain Yield Grain yield/plot (Shell weight)

TABLE 14 Timing Evaluation Description V8-V12 Chlorophyll V12-VT Earleaf area V15-15DAP Chl fluorescence V15-15DAP CER 15-25 DAPCarbohydrates sucrose, starch Pre-Harvest 1st internode diameterPre-Harvest Base 3 internode diameter Pre-Harvest Ear internode diameterMaturity Ear traits diameter, length, kernel number, kernel weight

Electron transport rates (ETR) and CO2 exchange rates (CER): ETR and CERare measured with Li6400LCF (Licor, Lincoln, Nebr.) around V9-R1 stages.Leaf chlorophyll fluorescence is a quick way to monitor the sourceactivity and is reported to be highly correlated with CO₂ assimilationunder varies conditions (Photosyn Research, 37: 89-102). The youngestfully expanded leaf or 2 leaves above the ear leaf is measured withactinic light 1500 (with 10% blue light) micromol m⁻² s⁻¹, 28° C., CO2levels 450 ppm. Ten plants are measured in each event. There are 2readings for each plant.

A hand-held chlorophyll meter SPAD-502 (Minolta-Japan) is used tomeasure the total chlorophyll level on live transgenic plants and thewild type counterparts a. Three trifoliates from each plant areanalyzed, and each trifoliate were analyzed three times. Then 9 datapoints are averaged to obtain the chlorophyll level. The number ofanalyzed plants of each genotype ranges from 5 to 8.

When selecting for yield improvement a useful statistical measurementapproach comprises three components, i.e. modeling spatialautocorrelation of the test field separately for each location,adjusting traits of recombinant DNA events for spatial dependence foreach location, and conducting an across location analysis. The firststep in modeling spatial autocorrelation is estimating the covarianceparameters of the semivariogram. A spherical covariance model is assumedto model the spatial autocorrelation. Because of the size and nature ofthe trial, it is likely that the spatial autocorrelation may change.Therefore, anisotropy is also assumed along with spherical covariancestructure. The following set of equations describes the statistical formof the anisotropic spherical covariance model.

${{C\left( {h;\theta} \right)} = {{{vI}\left( {h = 0} \right)} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < 1} \right)}}}},$

where I(•) is the indicator function, h=√{square root over ({dot over(x)}²+{dot over (y)}²)}, and

{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]ω_(x)

{dot over (y)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]ω_(y)

where s₁=(x₁, y₁) are the spatial coordinates of one location ands₂=(x₂, y₂) are the spatial coordinates of the second location. Thereare 5 covariance parameters, θ=(ν, σ², ρ, ω_(n), ω_(j)), where ν is thenugget effect, σ² is the partial sill, ρ is a rotation in degreesclockwise from north, ω_(n) is a scaling parameter for the minor axisand ω_(j) is a scaling parameter for the major axis of an anisotropicalellipse of equal covariance. The five covariance parameters that definesthe spatial trend will then be estimated by using data from heavilyreplicated pollinator plots via restricted maximum likelihood approach.In a multi-location field trial, spatial trend are modeled separatelyfor each location.

After obtaining the variance parameters of the model, avariance-covariance structure is generated for the data set to beanalyzed. This variance-covariance structure contains spatialinformation required to adjust yield data for spatial dependence. Inthis case, a nested model that best represents the treatment andexperimental design of the study is used along with thevariance-covariance structure to adjust the yield data. During thisprocess the nursery or the seed batch effects can also be modeled andestimated to adjust the yields for any yield parity caused by seed batchdifferences. After spatially adjusted data from different locations aregenerated, all adjusted data is combined and analyzed assuming locationsas replications. In this analysis, intra and inter-location variancesare combined to estimate the standard error of yield from transgenicplants and control plants. Relative mean comparisons are used toindicate statistically significant yield improvements.

C. Selection for Enhanced Water Use Efficiency (WUE)

Described in this example is a high-throughput method for greenhouseselection of transgenic corn plants to wild type corn plants (tested asinbreds or hybrids) for water use efficiency and method for selectiontransgenic cotton plants for water use efficiency. This selectionprocess imposes 3 drought/re-water cycles on plants over a total periodof 15 days after an initial stress free growth period of 11 days. Eachcycle consists of 5 days, with no water being applied for the first fourdays and a water quenching on the 5th day of the cycle. The primaryphenotypes analyzed by the selection method are the changes in plantgrowth rate as determined by height and biomass during a vegetativedrought treatment. The hydration status of the shoot tissues followingthe drought is also measured. The plant height is measured at three timepoints. The first is taken just prior to the onset drought when theplant is 11 days old, which is the shoot initial height (SIH). The plantheight is also measured halfway throughout the drought/re-water regimen,on day 18 after planting, to give rise to the shoot mid-drought height(SMH). Upon the completion of the final drought cycle on day 26 afterplanting, the shoot portion of the plant is harvested and measured for afinal height, which is the shoot wilt height (SWH) and also measured forshoot wilted biomass (SWM). The shoot is placed in water at 40 degreeCelsius in the dark. Three days later, the shoot is weighted to giverise to the shoot turgid weight (STM). After drying in an oven for fourdays, the shoots are weighted for shoot dry biomass (SDM). The shootaverage height (SAH) is the mean plant height across the 3 heightmeasurements. The procedure described above may be adjusted for +/−˜oneday for each step given the situation.

To correct for slight differences between plants, a size correctedgrowth value is derived from SIH and SWH. This is the Relative GrowthRate (RGR). Relative Growth Rate (RGR) is calculated for each shootusing the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)*100]. Relative watercontent (RWC) is a measurement of how much (%) of the plant was water atharvest. Water Content (RWC) is calculated for each shoot using theformula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants ofthis age run around 98% RWC.

Progeny transgenic plants are selected from a population of transgeniccotton events under specified growing conditions and are compared withcontrol cotton plants. Control cotton plants are substantially the samecotton genotype but without the recombinant DNA, for example, either aparental cotton plant of the same genotype that was not transformed withthe identical recombinant DNA or a negative isoline of the transformedplant. Additionally, a commercial cotton cultivar adapted to thegeographical region and cultivation conditions, i.e. cotton varietyST474, cotton variety FM 958, and cotton variety Siokra L-23, are usedto compare the relative performance of the transgenic cotton plantscontaining the recombinant DNA. The specified culture conditions aregrowing a first set of transgenic and control plants under “wet”conditions, i.e. irrigated in the range of 85 to 100 percent ofevapotranspiration to provide leaf water potential of −14 to −18 bars,and growing a second set of transgenic and control plants under “dry”conditions, i.e. irrigated in the range of 40 to 60 percent ofevapotranspiration to provide a leaf water potential of −21 to −25 bars.Pest control, such as weed and insect control is applied equally to bothwet and dry treatments as needed. Data gathered during the trialincludes weather records throughout the growing season includingdetailed records of rainfall; soil characterization information; anyherbicide or insecticide applications; any gross agronomic differencesobserved such as leaf morphology, branching habit, leaf color, time toflowering, and fruiting pattern; plant height at various points duringthe trial; stand density; node and fruit number including node abovewhite flower and node above crack boll measurements; and visual wiltscoring. Cotton boll samples are taken and analyzed for lint fractionand fiber quality. The cotton is harvested at the normal harvesttimeframe for the trial area. Enhanced water use efficiency is indicatedby increased yield, improved relative water content, enhanced leaf waterpotential, increased biomass, enhanced leaf extension rates, andimproved fiber parameters.

D. Selection for Growth Under Cold Stress

(1) Cold germination assay—Three sets of seeds are used for the assay.The first set consists of positive transgenic events (F1 hybrid) wherethe genes of the present invention are expressed in the seed. The secondseed set is nontransgenic, wild-type negative control made from the samegenotype as the transgenic events. The third set consisted of two coldtolerant and one cold sensitive commercial check lines of corn. Allseeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide,Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan isapplied per 45 g of corn seeds by mixing it well and drying thefungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within anindividual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seedsfrom an event are placed into one cell of the germination tray. Eachtray can hold 21 transgenic events and 3 replicates of wildtype(LH244SDms+LH59), which is randomized in a complete block design. Forevery event there are five replications (five trays). The trays areplaced at 9.7 C for 24 days (no light) in a Convrion growth chamber(Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Twohundred and fifty millilters of deionized water are added to eachgermination tray. Germination counts are taken 10th, 11th, 12th, 13th,14th, 17th, 19th, 21st, and 24th day after start date of the experiment.Seeds are considered germinated if the emerged radicle size is 1 cm.From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1−n _(i) ]*[P _(i) −P _(i−1)]))/T

Where T is the total number of days for which the germination assay isperformed. The number of days after planting is defined by n. “i”indicated the number of times the germination had been counted,including the current day. P is the percentage of seeds germinatedduring any given rating. Statistical differences are calculated betweentransgenic events and wild type control. After statistical analysis, theevents that show a statistical significance at the p level of less than0.1 relative to wild-type controls will advance to a secondary coldselection. The secondary cold screen is conducted in the same manner ofthe primary selection only increasing the number of repetitions to ten.Statistical analysis of the data from the secondary selection isconducted to identify the events that show a statistical significance atthe p level of less than 0.05 relative to wild-type controls.

TABLE 15 Germination Index 1st Run 2nd Run PEP SEQ % P % P ID CONSTRUCTEvent Change value Change value 266 PMON92840 MON810, ZM_M106115 280.079 15 0.233 PMON92840 MON810, ZM_M107208 58 0.000 24 0.049 PMON92840MON810, ZM_M107212 36 0.026 34 0.006 PMON92840 MON810, ZM_M107214 530.001 26 0.035 PMON92840 MON810, ZM_M107221 29 0.072 −5 0.663 PMON92840MON810, ZM_M107224 60 0.000 35 0.004 PMON92840 MON810, ZM_M107228 390.017 30 0.016 284 PMON92854 MON810, ZM_M103991 28 0.070 9 0.364PMON92854 MON810, ZM_M104002 35 0.025 8 0.412 PMON92854 MON810,ZM_M105195 27 0.082 10 0.321 PMON92854 MON810, ZM_M105213 30 0.060 210.033 PMON92854 MON810, ZM_M105218 74 0.000 49 0.000 PMON92854 MON810,ZM_M105267 43 0.006 28 0.004 PMON92854 MON810, ZM_M106123 −1 0.965 300.002

(2) Cold Shock assay—The experimental set-up for the cold shock assay isthe same as described in the above cold germination assay except seedswere grown in potted media for the cold shock assay.

The desired numbers of 2.5″ square plastic pots are placed on flats(n=32, 4×8). Pots were filled with Metro Mix 200 soil-less mediacontaining 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots andFlat are obtained from Hummert International, Earth City, Mo.). Afterplanting seeds, pots are placed in a growth chamber set at 23° C.,relative humidity of 65% with 12 hour day and night photoperiod (300uE/m2-min) Planted seeds are watered for 20 minute every other day bysub-irrigation and flats were rotated every third day in a growthchamber for growing corn seedlings.

On the 10^(th) day after planting the transgenic positive and wild-typenegative (WT) plants are positioned in flats in an alternating pattern.Chlorophyll fluorescence of plants is measured on the 10^(th) day duringthe dark period of growth by using a PAM-2000 portable fluorometer asper the manufacturer's instructions (Walz, Germany). After chlorophyllmeasurements, leaf samples from each event are collected for confirmingthe expression of genes of the present invention. For expressionanalysis six V1 leaf tips from each selection are randomly harvested.The flats are moved to a growth chamber set at 5° C. All otherconditions such as humidity, day/night cycle and light intensity areheld constant in the growth chamber. The flats are sub-irrigated everyday after transfer to the cold temperature. On the 4^(th) daychlorophyll fluorescence is measured. Plants are transferred to normalgrowth conditions after six days of cold shock treatment and allowed torecover for the next three days. During this recovery period the lengthof the V3 leaf is measured on the 1^(st) and 3^(th) days. After two daysof recovery V2 leaf damage is determined visually by estimating percentof green V2 leaf.

Statistical differences in V3 leaf growth, V2 leaf necrosis andfluorescence during pre-shock and cold shock can be used for estimationof cold shock damage on corn plants.

(3) Early seedling growth assay—Three sets of seeds are used for theexperiment. The first set consists of positive transgenic events (F1hybrid) where the genes of the present invention are expressed in theseed. The second seed set is nontransgenic, wild-type negative controlmade from the same genotype as the transgenic events. The third seed setconsists of two cold tolerant and two cold sensitive commercial checklines of corn. All seeds are treated with a fungicide “Captan”,(3a,4,7,a-tetrahydro-2-Rtrichloromethly)thiol-1H-isoindole-1,3(2H)-dione,Drex Chemical Co. Memphis, Tenn.). Captan (0.43 mL) was applied per 45 gof corn seeds by mixing it well and drying the fungicide prior to theexperiment.

Seeds are grown in germination paper for the early seedling growthassay. Three 12″−18″ pieces of germination paper (Anchor Paper #SD7606)are used for each entry in the test (three repetitions per transgenicevent). The papers are wetted in a solution of 0.5% KNO₃ and 0.1%Thyram.

For each paper fifteen seeds are placed on the line evenly spaced downthe length of the paper. The fifteen seeds are positioned on the papersuch that the radical would grow downward, for example longer distanceto the paper's edge. The wet paper is rolled up starting from one of theshort ends. The paper is rolled evenly and tight enough to hold theseeds in place. The roll is secured into place with two large paperclips, one at the top and one at the bottom. The rolls are incubated ina growth chamber at 23° C. for three days in a randomized complete blockdesign within an appropriate container. The chamber is set for 65%humidity with no light cycle. For the cold stress treatment the rollsare then incubated in a growth chamber at 12° C. for twelve days. Thechamber is set for 65% humidity with no light cycle.

After the cold treatment the germination papers are unrolled and theseeds that did not germinate are discarded. The lengths of the radicleand coleoptile for each seed are measured through an automated imagingprogram that automatically collects and processes the images. Theimaging program automatically measures the shoot length, root length,and whole seedling length of every individual seedling and thencalculates the average of each roll.

After statistical analysis, the events that show a statisticalsignificance at the p level of less than 0.1 relative to wild-typecontrols will advance to a secondary cold selection. The secondary coldselection is conducted in the same manner of the primary selection onlyincreasing the number of repetitions to five. Statistical analysis ofthe data from the secondary selection is conducted to identify theevents that show a statistical significance at the p level of less than0.05 relative to wild-type controls.

(4). Cold Field Efficacy Trial

This example sets forth a cold field efficacy trial to identify geneconstructs that confer enhanced cold vigor at germination and earlyseedling growth under early spring planting field conditions inconventional-till and simulated no-till environments. Seeds are plantedinto the ground around two weeks before local farmers are beginning toplant corn so that a significant cold stress is exerted onto the crop,named as cold treatment. Seeds also are planted under local optimalplanting conditions such that the crop has little or no exposure to coldcondition, named as normal treatment. The cold field efficacy trials arecarried out in five locations, including Glyndon Minn., Mason Mich.,Monmouth Ill., Dayton Iowa, Mystic Conn. At each location, seeds areplanted under both cold and normal conditions with 3 repetitions pertreatment, 20 kernels per row and single row per plot. Seeds are planted1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperaturemonitors are set up at each location to monitor both air and soiltemperature daily.

Seed emergence is defined as the point when the growing shoot breaks thesoil surface. The number of emerged seedling in each plot is countedeveryday from the day the earliest plot begins to emerge until nosignificant changes in emergence occur. In addition, for each plantingdate, the latest date when emergence is 0 in all plots is also recorded.Seedling vigor is also rated at V3-V4 stage before the average of cornplant height reaches 10 inches, with 1=excellent early growth, 5=Averagegrowth and 9=poor growth. Days to 50% emergence, maximum percentemergence and seedling vigor are calculated using SAS software for thedata within each location or across all locations.

E. Screens for Transgenic Plant Seeds with Increased Protein and/or OilLevels

This example sets forth a high-throughput selection for identifyingplant seeds with improvement in seed composition using the Infratec 1200series Grain Analyzer, which is a near-infrared transmittancespectrometer used to determine the composition of a bulk seed sample.Near infrared analysis is a non-destructive, high-throughput method thatcan analyze multiple traits in a single sample scan. An NIR calibrationfor the analytes of interest is used to predict the values of an unknownsample. The NIR spectrum is obtained for the sample and compared to thecalibration using a complex chemometric software package that provides apredicted values as well as information on how well the sample fits inthe calibration.

Infratec Model 1221, 1225, or 1227 with transport module by Foss NorthAmerica is used with cuvette, item #1000-4033, Foss North America or forsmall samples with small cell cuvette, Foss standard cuvette modified byLeon Girard Co. Corn and soy check samples of varying compositionmaintained in check cell cuvettes are supplied by Leon Girard Co. NITcollection software is provided by Maximum Consulting Inc. Software.Calculations are performed automatically by the software. Seed samplesare received in packets or containers with barcode labels from thecustomer. The seed is poured into the cuvettes and analyzed as received.

TABLE 16 Typical sample(s): Whole grain corn and soybean seedsAnalytical time to run method: Less than 0.75 min per sample Totalelapsed time per run: 1.5 minute per sample Typical and minimum samplesize: Corn typical: 50 cc; minimum 30 cc Soybean typical: 50 cc; minimum5 cc Typical analytical range: Determined in part by the specificcalibration. Corn-moisture 5-15%, oil 5-20%, protein 5-30%, starch50-75%, and density 1.0-1.3%. Soybean-moisture 5-15%, oil 15- 25%, andprotein 35-50%.

TABLE 17 Transgenic corn plants have an increased oil level in seeds2004 Data 2005 Data PEP SEQ Oil P Oil P ID NO Event Construct Deltavalue delta value 231 ZM_S90572 PMON17730 0.18 0.22 0.25 0.04 ZM_S90588PMON17730 N/A N/A 0.10 0.33 ZM_S90610 PMON17730 N/A N/A −0.03 0.78ZM_S90614 PMON17730 0.26 0.08 0.47 0.00 ZM_S90622 PMON17730 −0.03  0.820.31 0.00

TABLE 18 Transgenic corn plants have an increased protein level in seeds1st Inbred protein trial 2nd Inbred protein trial PEP SEQ Mean Mean MeanMean ID NO Construct Event transgenic control Delta Pvalue transgeniccontrol Delta Pvalue 208 PMON92607 ZM_M106133 14.06 10.39 3.67 0 11.9110.06 1.84 0.0023 ZM_M106129 16.46 10.39 6.07 0 16.00 10.06 5.94 0ZM_M105269 15.80 10.39 5.41 0 14.89 10.06 4.83 0 ZM_M105268 14.07 10.393.67 0 12.81 10.06 2.74 0 ZM_M104742 12.57 10.39 2.18 0.0064 12.52 10.062.46 0 ZM_M104740 14.45 10.39 4.06 0 12.93 10.06 2.86 0 ZM_M104403 12.9010.39 2.51 0.0017 12.60 10.06 2.53 0 ZM_M104399 13.21 10.39 2.82 0.000414.04 10.06 3.98 0 ZM_M104398 14.91 10.39 4.51 0 12.79 10.06 2.73 0ZM_M104396 12.13 10.39 1.74 0.0289 12.90 10.06 2.83 0 ZM_M104385 13.1210.39 2.72 0.0007 12.89 10.06 2.82 0 ZM_M104371 12.23 10.39 1.83 0.021312.18 10.06 2.12 0.0004 ZM_M104369 13.41 10.39 3.01 0.0002 11.35 10.061.29 0.0309 ZM_M103621 12.26 10.39 1.86 0.0191 10.76 10.06 0.69 0.2425ZM_M106138 13.74 10.39 3.35 0 13.34 10.06 3.28 0

TABLE 19 Transgenic soybean plants have an increased seed oil level seedoil content seed protein content PEP SEQ control transgenic controltransgenic ID NO construct event run mean mean delta mean mean delta 231PMON94697 construct 1 20.0 19.7 −0.3 42.0 42.9 0.9 analysis 2 19.6 20.20.6* 43.3 43.6 0.3 3 19.9 20.6 0.7* 42.5 43.1 0.6 GM_A79833 1 20.2 19.7−0.3 42.0 42.9 0.9 2 19.6 19.9 0.3 43.3 43.5 0.2 GM_A79838 2 19.6 19.80.2 43.3 45.2 1.9* GM_A79839 2 19.6 20.4 0.8* 43.3 43.7 0.4 GM_A79857 219.6 19.9 0.3 43.3 43.7 0.4 GM_A79859 2 19.6 20.6 1.0* 43.3 43.7 −0.6GM_A79894 2 19.6 20.4 0.8* 43.3 43.1 −0.2 GM_A79896 2 19.6 19.8 0.2 43.344.4 1.1* GM_A79914 2 19.6 20.9 1.3* 43.3 42.7 −0.6 3 19.9 20.6 0.7 42.543.4 0.9 GM_A79934 3 19.9 20.3 0.4* 42.5 43.3 0.8* GM_A79936 3 19.9 21.01.1* 42.5 42.5 0.0 Data point with “*”indicate a statisticallysignificant delta (the difference between transgenic and controlplants). Seed protein or oil is measured as a percentage of total seedcomposition.

1. (canceled)
 2. A plant cell with stably integrated, recombinant DNAcomprising a promoter that is functional in plant cells and that isoperably linked to DNA from a plant, bacteria or yeast that encodes aprotein having at least one domain of amino acids in a sequence thatexceeds the Pfam gathering cutoff for amino acid sequence alignment witha protein domain family identified by a Pfam name wherein the Pfamgathering cutoff for said protein domain families is 15; wherein saidplant cell is selected from a population of plant cells with saidrecombinant DNA by screening plants that are regenerated from plantcells in said population and that express said protein for an enhancedtrait as compared to control plants that do not have said recombinantDNA; and wherein said enhanced trait is enhanced water use efficiency,or increased yield.
 3. A plant cell of claim 2 wherein said protein hasan amino acid sequence with at least 90% identity to SEQ ID NO:
 249. 4.A plant cell of claim 2 wherein said protein is phycocyanin alphaphycocyanobilin lyase.
 5. A plant cell of claim 2 further comprising DNAexpressing a protein that provides tolerance from exposure to anherbicide applied at levels that are lethal to a wild type of said plantcell.
 6. A plant cell of claim 5 wherein the agent of said herbicide isa glyphosate, dicamba, or glufosinate compound.
 7. A transgenic plantcomprising a plurality of the plant cell of claim 2
 8. A transgenicplant of claim 7 which is homozygous for said recombinant DNA.
 9. Atransgenic seed comprising a plurality of the plant cell of claim
 2. 10.A transgenic seed of claim 9 from a corn, soybean, cotton, canola,alfalfa, wheat or rice plant.
 11. A transgenic pollen grain comprising ahaploid derivative of a plant cell of claim
 2. 12. A method formanufacturing non-natural, transgenic seed that can be used to produce acrop of transgenic plants with an enhanced trait resulting fromexpression of stably-integrated, recombinant DNA comprising a promoterthat is (a) functional in plant cells and (b) is operably linked to DNAfrom a plant, bacteria or yeast that encodes a protein having at leastone domain of amino acids in a sequence that exceeds the Pfam gatheringcutoff for amino acid sequence alignment with a protein domain familyidentified by a Pfam name; wherein the gathering cutoff for said proteindomain families is 15; and wherein said enhanced trait is enhanced wateruse efficiency or increased yield, said method for manufacturing saidseed comprising: (a) screening a population of plants for said enhancedtrait and said recombinant DNA, wherein individual plants in saidpopulation can exhibit said trait at a level less than, essentially thesame as or greater than the level that said trait is exhibited incontrol plants which do not express the recombinant DNA, (b) selectingfrom said population one or more plants that exhibit the trait at alevel greater than the level that said trait is exhibited in controlplants, (c) verifying that said recombinant DNA is stably integrated insaid selected plants, (d) analyzing tissue of a selected plant todetermine the production of a protein having the function of a proteinencoded by nucleotides in a sequence of SEQ ID NO: 56; and (e)collecting seed from a selected plant.
 13. A method of claim 12 whereinplants in said population further comprise DNA expressing a protein thatprovides tolerance to exposure to an herbicide applied at levels thatare lethal to wild type plant cells, and wherein said selecting iseffected by treating said population with said herbicide.
 14. A methodof claim 13 wherein said herbicide comprises a glyphosate, dicamba, orglufosinate compound.
 15. A method of claim 12 wherein said selecting iseffected by identifying plants with said enhanced trait.
 16. A method ofclaim 12 wherein said seed is corn, soybean, cotton, alfalfa, wheat orrice seed.
 17. A method of producing hybrid corn seed comprising:acquiring hybrid corn seed from a herbicide tolerant corn plant whichalso has stably-integrated, recombinant DNA comprising a promoter thatis (a) functional in plant cells and (b) is operably linked to DNA thatencodes a protein having at least one domain of amino acids in asequence that exceeds the Pfam gathering cutoff for amino acid sequencealignment with a protein domain family identified by a Pfam name; (a)wherein the gathering cutoff for said protein domain families is 15; (b)producing corn plants from said hybrid corn seed, wherein a fraction ofthe plants produced from said hybrid corn seed is homozygous for saidrecombinant DNA, a fraction of the plants produced from said hybrid cornseed is hemizygous for said recombinant DNA, and a fraction of theplants produced from said hybrid corn seed has none of said recombinantDNA; (c) selecting corn plants which are homozygous and hemizygous forsaid recombinant DNA by treating with an herbicide; (d) collecting seedfrom herbicide-treated-surviving corn plants and planting said seed toproduce further progeny corn plants; (e) repeating steps (c) and (d) atleast once to produce an inbred corn line; (f) crossing said inbred cornline with a second corn line to produce hybrid seed.
 18. A method ofselecting a plant comprising cells of claim 2 wherein an immunoreactiveantibody is used to detect the presence of said protein in seed or planttissue.
 19. Anti-counterfeit milled seed having, as an indication oforigin, a plant cell of claim
 2. 20. A method of growing a corn, cottonor soybean crop without irrigation water comprising planting seed havingplant cells of claim 2 which are selected for enhanced water useefficiency.